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Review of molecular methods for medical microbiology testing.

History

Since the conception of the polymerase chain reaction (PCR) in 1983 by Dr Kary Mullis (and others from the Cetus Corporation) and the subsequent publication in Science in 1985, this technique has been utilised in many forms in order to produce virtually unlimited copies of genetic material in the laboratory (amplification). Amplification of as little as a single strand of DNA target sequence enables PCR to detect extremely low concentrations of nucleic acid. This technology has reduced the reliance of the clinical microbiology laboratory on culture-based methods and created new opportunities for the clinical laboratory to more efficiently affect patient care (1).

In 1990 Roche Diagnostics purchased the patents related to PCR, imposing license conditions on subsequent users of the methodology. The techniques for these processes have been documented by Roche Diagnostics (2-4).

The use of a range of molecular tools allows highly sensitive and specific, culture-independent detection of infectious agents in clinical specimens (5). An example of the sensitivity obtained by PCR is where Chamydia spp. can be detected to a single target in a 1012-fold background of unrelated DNA. An optimised PCR can be at least 150-fold, but more often about 1000-fold more sensitive than the cell culture (6).

This new technology evolved at a time when enzyme immunoassays for viral detection particularly, had reached the limit of their sensitivity (7). Of all fields in medical microbiology, detection by PCR has had the strongest impact on virology where standard microbiological diagnosis by culture requires much greater effort than for many other pathogens (6). For safety reasons molecular techniques, rather than culture, are the most advisable for the detection of highly infectious viruses, e.g. avian influenza (H5N1) and SARS etc.

In the case of medical microbiology, molecular methods also enable the detection of pathogens that are difficult to cultivate, or are slow growing or unculturable (1). Nucleic acids (DNA and RNA) present in infectious organisms can be amplified. The genetic material of many viruses is RNA while DNA is the genetic material in bacteria and other viruses.

Since the original description of PCR, a number of modifications, advances and alternative systems for in-vitro amplification of nucleic acids have been developed to meet various requirements for improved detection of DNA and RNA. Some of these different applications for PCR will be discussed below with reference to both the detection of bacteria and viruses.

The genome of each organism contains highly conserved, coded regions that are common to that type of organism and variable regions that result in individual traits. Primers are usually selected from the highly conserved region of the genetic code specific for the target organism (8).

Principle of amplification

Amplification is based on complementarity between a target nucleic acid sequence of interest, present in infectious agents, and primers specifically designed to bind (hybridise/anneal) only to a region within that target nucleic acid. The complementary nature of the primer-target binding gives the assay specificity (sensitivity is due the amplification of a small number of target sequences as explained below). One strand of DNA runs in the 5 prime (5') to 3 prime (3') direction and the complementary strand runs in the 3' to 5' direction. Primers are designed in the 5' to 3' direction and therefore bind to the two strands of double stranded DNA of the target sequence in opposite directions. Primers are used to initiate new DNA synthesis by heat-stable Taq DNA polymerase (derived originally from the hot-spring bacterium Thermus aquaticus but now usually produced by recombinant DNA technology.

Nucleic acid primers or probes can distinguish minute differences in genetic sequence. Optimal conditions during amplification allow complementary binding (hybridisation) only if both sequences match exactly. The primers determine the specificity and size of the amplified product. When the primers anneal, Taq DNA polymerase, using the DNA building blocks dinucleotide triphosphates (dNTPs) as substrates, initiates replication of the target sequence from the 3' ends of the primers. Repeated cycles of amplification of extracted nucleic acid with specific forward and reverse primers is carried out on a thermocycler resulting in an exponential increase in the initial number of strands of DNA. The exponential amplification of the target sequence gives the PCR assay great sensitivity.

Application of PCR in microbiology

Organisms that are non-viable, slow growing or that cannot be isolated or detected in standard culture systems can all be detected with increased sensitivity and specificity within a suitable time frame by PCR. It enables quantification of the organism as well as detection of antimicrobial resistance in organisms such as methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus spp. (VRE) (9,10) and Mycobacterium tuberculosis (6).

It is imperative that the PCR assay be carefully optimised and validated before introduction into the routine molecular microbiology laboratory. This process should produce a robust assay that has all chemicals at optimal concentrations and the most appropriate thermal reaction parameters. Typically some DNA or RNA sequence of the organism is known (for primer design) otherwise 'universal' primers (e.g. to the 16S region of bacteria) may be used, followed by DNA sequencing to confirm the organism's identity. For amplification of DNA there must be at least one intact copy of the target DNA/RNA present in the sample.

Extraction of DNA/RNA (commercial columns)

Only a small amount of clinical specimen is required. DNA/RNA template quality is very important--especially in the newer forms of PCR that use fluorescent probes. Typically small centrifuge columns containing a DNA/RNA-binding matrix are used in the clinical laboratory rather than the historical organic solvent methods.

The column extraction methods comprise the following basic steps: Step 1. Lysis

Lysis buffer often containing guanidine thiocyanate or similar plus a detergent such as Triton-X or SDS lyses the cells releasing the nucleic acids. Proteinase K enzyme for protease digestion is also added and rapidly inactivates endogenous nucleases such as RNAses and DNAses.

Step 2. Precipitation

The lysate is then mixed with alcohol to precipitate the DNA and then loaded onto the extraction column.

Step 3. Binding to membrane/washing

DNA is absorbed onto the silica-gel membrane. DNA bound to the membrane is purified by washing with a high salt buffer to ensure complete removal of any residual contaminants such as salts, proteins, cellular components etc.

Step 4. Elution of purified DNA/RNA

Purified DNA/RNA is eluted from the extraction column with a low salt buffer or water. The eluate is then ready for PCR testing with the reaction mix.

Reaction mix components

The reaction mix consists of buffer, [Mg.sup.2+], dNTPs, primers, Taq polymerase enzyme, and water. These components are essential for efficient replication of the target nucleic acid to occur.

Buffer provides the optimal pH and ionic strength for polymerase activity.

Mg[Cl.sub.2] forms soluble complexes with dNTPs to produce the actual substrate that the Taq polymerase recognises. [Mg.sup.2+] influences enzyme activity and increases the melting temperature ([T.sub.m]) of double stranded DNA (dsDNA).

dNTPs are required as a supply of individual nucleotides (Cytosine, Adenine, Thymine, Guanine--C, A, T, G) and may be supplied as a sets of four separate deoxynucleotides (dCTP, dATP, dTTP, dGTP) or as a pre-mixed solution. The concentration of Mg[Cl.sub.2] and dNTPs must be in the correct ratio for optimum performance since the dNTPs will bind Mg[Cl.sub.2].

Oligonucleotide primers contain sequences that are complementary to specific sequences that flank the target sequence of the DNA that is to be amplified. The sequence and concentration of primers determines the overall assay success. Well selected primers increase reaction specificity by ensuring that they bind only to the desired target sequence to be amplified. Stringency of primer hybridisation can be increased by higher annealing temperatures, lower Mg[Cl.sub.2] concentrations, optimal concentrations of enzyme and primers, annealing time, extension time and the number of PCR cycles.

Taq DNA polymerase is required to synthesise new dsDNA, beginning at the 3' end of the primer, and facilitates the binding and joining of the complementary nucleotides (Taq DNA polymerase does not recognise the end of the target sequence and will continue extending original DNA past the region of interest). Newer Taq polymerases (employing "hot start" methods) are thermostable and only activated after the initial denaturation step in PCR cycling at 94[degrees]C thereby providing an automatic "hot start" (6). These newer Taq polymerases remain inactive during PCR set-up. Hot start enzymes cannot elongate nonspecific primer-template hybrids that may form at lower temperatures, which increases sensitivity, specificity and yield while allowing assembly of reactions at room temperature.

Water is included to make up the reaction volume.

Thermocycling

The programmed thermocycler mimics the process of DNA replication. PCR is a three-step process, referred to as a cycle, that is repeated a specified number of times One PCR cycle consists of:

Step 1. Denaturation

The reaction mix is heated to >90[degrees]C where all double-stranded DNA is separated into single strands. Taq polymerase is not denatured during the high heat denaturation steps.

Step 2. Annealing

The temperature is dropped to between 55-65[degrees]C, enabling the forward primer to bind specifically to one target DNA strand and the reverse primer to bind specifically to the complementary target DNA strand. The annealing temperature or Tm varies for different assays and is determined by the melting temperature of the primers and is the temperature at which 50% of the oligonucleotide-target duplexes have formed. [approximately equal to]

Step 3. Primer extension

The temperature is raised to [approximately equal to] 72[degrees]C for optimal activity of the Taq DNA polymerase enzyme. Taq DNA polymerase catalyses primer extension as complementary nucleotides (supplied from the dNTPs) are incorporated, synthesising new dsDNA complementary to the original double stranded target DNA region. Taq DNA polymerase can add approximately 60 bases/second at 72[degrees]C.

At the end of the first PCR cycle there is double the number of new DNA strands identical to the original target. This new dsDNA then serves as a template for the synthesis of new DNA in the next cycle. With each new cycle the number of synthesised DNA increases exponentially. This is the CHAIN REACTION in the PCR method. This cyclical process is repeated up to [approximately equal to] 40 times to amplify the original DNA in an exponential fashion to a product that is easily detectable on a gel (endpoint analysis). Conventional amplification time is [approximately equal to] 2-3 hours.

Reverse transcription PCR (RT-PCR)

PCR usually only detects DNA, as RNA is not an efficient substrate for Taq DNA polymerase. Therefore, for RNA, it is necessary to perform reverse transcription, using a reverse transcriptase enzyme, before initiating PCR amplification (RT-PCR). (Some enzymes do have both reverse transcriptases and DNA polymerases.) Reverse transcription produces a copy of the RNA strand, as complementary DNA (first strand cDNA) in the presence of [Mg.sup.2+] ions, high concentrations of dNTPs and primers. These primers may be either random (e.g. 6 bases), a stretch of T's, a sequence specific for the organism of interest or a combination of these. The reverse transcription process includes extracted RNA, buffer containing [Mg.sup.2+], RT enzyme, site specific primer (that selectively primes the RNA of interest), RNAse inhibitor (that disrupts cells and inactivates RNAses) and dNTPs.

The RT-PCR process consists of:

Step 1. Annealing of the primer(s) to the single stranded RNA.

Step 2. RT enzyme catalysing primer extension to create first strand cDNA.

Step 3. The resulting first strand cDNA with PCR premix is then available for amplification.

Nested PCR

Nested PCR has the ability to increase both the sensitivity and specificity of PCR, by using two sets of primers an "external" and an "internal" set and two rounds of amplification. There is an increase in the sensitivity from increased total cycle number. Increased specificity is obtained from annealing of the second inner primer set to sequences only amplified from the first PCR. However one disadvantage of nested PCR is that when the second round amplification is set up there is the potential for contamination (1). A variation of nested PCR is a hemi-nested PCR where the second primer set involves an internal primer and one of the external primers.

Multiplex PCR

PCR assays utilise two or more primer sets in the same reaction, this enables simultaneous co-amplification of different nucleic acid targets. Multiplexing can only work when the same set of amplification conditions are suitable for all primers and the primers don't anneal to each other. Additionally the products formed must differ adequately to enable accurate interpretation of the results. However this method is often less sensitive than PCRs with single primer sets due to competition between targets to be amplified (11).

Internal controls

Controls can be added to the PCR amplification mix to detect inhibition of PCR amplification and as a control for the correct performance of the specimen preparation and amplification steps. Internal controls are used to monitor variability within a PCR assay attributed to differences in reaction components, PCR inhibitors, operator-to-operator variance and instrumentation performance. The absence of amplification of an internal control due to inhibitors could also indicate prevention of amplification of the desired organism, so that false negative results could be obtained. However, if the internal controls are added directly to the PCR mix then the assay is, essentially, a multiplex assay. Alternatively a reference gene that is found in all human nucleated cells such as [beta] globin can be assayed separately for the presence of inhibitors.

Limitations of PCR Inhibitors

The presence of inhibitors in clinical specimens such as heme, acidic polysaccharides, nucleases, EDTA, SDS and guanidine HCl have all been demonstrated to inhibit PCR (12), by interfering with the action of DNA polymerase (6). An internal control (e.g. an artificial construct or a reference gene in the sample) should be incorporated into the testing specimen to monitor inhibition of the PCR by such substances that may be present in the patient's specimen (9).

Antimicrobial therapy--where treatment has been successful or alternatively, the patient's own defences may have killed the organism. PCR can still detect non-viable organisms (28) which may no longer be causing infection.

Relevance of result

PCR may be too sensitive for some applications, detecting the nucleic acid of an organism that is present at non-pathogenic levels (13).

Contamination

Cross contamination can easily occur unless a careful work ethic is followed. Stringent adherence to separated work places for the following must be observed:

1. Specimen preparation.

2. Setting up PCR reactions.

3. Post-PCR analysis.

Within each of these work areas the following good laboratory practice should be employed:

1. Keep laboratory areas clean.

2. Use aseptic technique, gloves, dedicated equipment, new/sterile equipment, using positive displacement pipettes with barrier tips, avoid creating aerosols when working.

3. Avoid cross-contamination between samples.

4. Avoid cross-contamination from previous samples.

5. Include control reactions in every run to monitor the test.

A further method that can limit contamination is where the amplicon can be inactivated by incorporating deoxyuridine (dUTP) instead of dTTP in the PCR assay, so that any amplicons that may contaminate subsequent assays are cleaved by uracil-N-glycosylase (9). However this is of limited use unless the assay is established with dUTP i.e before the contamination issue occurs.

Amplicon detection (fragment size analysis) for conventional PCR

Conventional PCR requires end point analysis of the amplified product. Detection of amplicons is by gel electrophoresis to determine fragment size. Electrophoresis refers to the migration of charged molecules through a liquid or gel medium when subjected to an electrical field. Migration rate through a gel matrix depends on several factors--net charge on a molecule at the pH at which the assay is performed, size and shape of molecule, electrical strength of voltage drop, pore size of gel and temperature. The charge in an electrical field moves analytes through the gel (14).

Amplicon detection--components Agarose gel

The concentration of agarose can be varied, controlling gel pore size to make possible the separation of nucleic acid molecules of a wide range of sizes. Migration of nucleic acid in agarose is affected by buffer used and voltage applied. The higher the concentration of agarose, the smaller the pore size. An agarose gel concentration of 2-4% separates fragments of 25-1000bp (14).

Polyacrylamide gel electrophoresis (PAGE)

For very high resolution of low-molecular weight DNA fragments, polyacrylamide gel is used as the pore size is typically much smaller than agarose (14). An alternative is NuSieve agarose which has higher resolution for visualising small DNA fragments (15).

Loading dye is included with the amplified product to be electrophoresed to increase the density of the sample allowing the amplicon to drop evenly into the well. The loading dye is a small molecule which migrates at a constant rate in an electrical field. This allows the progress of the smallest molecules in the gel to be visualised. The loading dye may be added during PCR reaction assembly or following the amplification process. Unlike ethidium bromide, the loading dye does not actually stain the DNA.

DNA gel stains

Ethidium bromide fluoresces in the presence of ss and ds DNA under long wavelength UV light. However, both the ethidium bromide and the UV light are mutagenic.

SYBR Safe[TM]

SYBR Safe[TM] binds to DNA and RNA, and was developed for reduced mutagenicity (16). Both the ethidium bromide and the SYBR Safe[TM] require a UV transilluminator for visualisation of DNA bands.

Molecular weight ladders (MW) are a mixture of DNA fragments, upon electrophoresis result in a regular pattern thus allowing for accurate sizing of DNA bands (amplicon).

EIA (ELISA)

EIA can also be used to detect digoxigenin-labeled amplified products. Products are obtained by using a PCR DIG labelling mix (dNTPs plus digoxigenin-labeled dUTP) in the PCR reaction. The labelled nucleotide is then incorporated into the PCR product as it is formed. The incorporated DIG label is detected using antidigoxigenin-alkaline phosphatase (AP) conjugate and colorimetric or XRay detection. This method increases the detection level where only small amounts of template DNA are available (enabling maximum yield of amplicon), by increasing the sensitivity of the assay by 10-100 fold over gel staining (11).

PCR purification (for sequencing)

Amplified DNA binds specifically to glass fibres in the purification spin columns in a similar way to template extraction systems. Bound DNA is purified by washing and spinning to remove contaminating primers, unincorporated nucleotides and salts. Purified DNA is eluted from the column using a low salt solution.

DNA sequencing (automated)

Amplified DNA product is used as a template which, along with suitable oligonucleotide primers and DNA polymerase, generates fragments that differ in length from each other by a single base. Fragments are separated by size, and bases at the end identified, recreating the original sequence. Various approaches are used to generate and detect the fragments.

One example is the Sanger dideoxy chain terminator method utilising dideoxy bases (ddNTPs) mixed with dNTPs. During replication a base is incorporated into a new chain and, if a ddNTP is incorporated, the replication reaction is terminated (17). A separate reaction is set up for each specific nucleotide with individual fluorescent labelled ddNTPs (in a lower ratio than dNTPs). DdNTPs are randomly added by DNA polymerase which in turn terminates elongation as a 3'-OH group is not present. Reaction products are separated by electrophoresis, which separates molecules according to size. The fluorescent base that terminated the PCR reaction is detected and the base identified.

The sequence obtained can be loaded into a Gene Bank e.g. PubMed, and the nearest sequence match obtained. If the appropriate DNA template has been selected, the genotype can be determined by sequencing. Genotyping may give an indication of virulence properties such as infectivity, virulence, antigenic variation and resistance to antiviral agents.

Quantitative PCR

The detection of fluorescence as an endpoint in real time PCR means target DNA present in the initial sample can be quantified. This is especially useful to monitor treatment. A series of standards with known amounts of target are amplified and a standard curve constructed (13).

Real Time PCR

This method utilises reaction components similar to traditional PCR and varies only in the detection process. Real Time PCR is no more sensitive than conventional PCR but is able to produce a result as the amplified product is being formed (18). The analysis of product formation is performed during the log phase of amplification producing a rapid result in a faster time-frame (Real Time) enabling rapid sample turnaround time. Additionally, real time PCR is performed in a closed system thus minimising the risk of contamination.

Real time PCR combines two instruments in one: a PCR thermal cycler and an integrated fluorescence detection device allowing for fluorescent monitoring to detect the formation of the PCR product. Automated amplification, detection and quantification (bacterial and viral loads) within the same closed system, allows for acceleration and increased efficiency of the process. The reaction time can be as short as 30 minutes (9).

For a commonly-found instrument in clinical laboratories, the LightCycler, reactions are performed in glass capillaries suspended in a chamber allowing a high surface-volume ratio of the PCR mix (9). These capillaries are heated by air allowing rapid, uniform heating producing a more rapid result. Fluorescent labelled probes bind to target DNA releasing fluorescence that is detected continuously by optical software. Fluorescence values versus cycle numbers are displayed. The increasing level of fluorescence is proportional to the number of target copies formed during the PCR process. The point at which fluorescence surpasses the noise threshold during amplification is called the threshold cycle or crossing point and is proportional to the number of amplified target copies present in the sample at that time (13).

Fluorescent signal can be detected by a number of systems such as:

--ethidium bromide, SYBR green

--hydrolysis probes, hybridisation probes

--molecular beacons, LUX probes

--scorpion primers

--Minor Groove Binding (MGB, Eclipse) probes.

Ethidium bromide and SYBR green dyes bind (intercalate) to any ds DNA and the increase in fluorescence is measured. However SYBR Green is more sensitive and specific than ethidium bromide (18). See Figure 1.

Sequence-specific probes are labelled with Fluorescence Resonance Energy Transfer (FRET) fluorophores. Fluorescence occurs due to a distance-dependent transfer of energy between two adjacent fluorophores and a fluorescent signal is only produced if the specific target is present. Alternatively, the energy may be transferred from a flourophore to a non-fluorescent compound and dissipated as heat, i.e. quenched, and in this case, fluorescence only occurs when the two compounds are separated (6). Descriptions of each type of probe follow.

Hydrolysis probes (Figure 2) e.g. "TaqMan[R] probes" (Roche Diagnostics), are labelled with a fluorescent reporter (R)(donor) and quencher (Q)(acceptor) in close proximity to each other. Specific binding to DNA target occurs, when DNA polymerase encounters the nonextendable probe, the probe is digested and R and Q are separated (the reporter is no longer quenched). The reporter can then emit fluorescence upon excitation (10). As TaqMan probes are hydrolysed they cannot be used for melting curve analysis.

Hybridisation probes (Figure 3) e.g. HybProbe[TM] hybridisation probes (Roche Diagnostics) comprise two fluorescent labelled, target-specific probes (Donor and Acceptor) that anneal to specific ssDNA. In close proximity the probes fluoresce due to FRET (10).

Molecular beacons and Scorpion[TM] primers also have a fluorescent reporter (R) and quencher (Q) in close proximity to each other. Sequence-specific binding results in an increase in the distance between R and Q to yield a detectable fluorescence (6).

LUX probes LUX[TM] (Light Upon eXtension) (Invitrogen). LUX[TM] primer sets include a self-quenched fluorogenic primer and a corresponding unlabeled primer creating a hairpin secondary structure which provides optimal quenching of the fluorophore. When dsDNA binds during PCR, the fluorophore is no longer quenched and fluorescence occurs (20).

Minor Groove Binding (MGB, Eclipse) probes

These dsDNA-binding agents are attached to the 3' end of TaqMan[R] probes to increase the [T.sub.m] value (by stabilization of hybridization). This process increases efficiency of reporter quenching (http://dorakmt. tripod.com/genetics/glosrt.html). Probes and primers can be made in many colours encompassing wavelengths from UV to infrared, making multiplex detection available (21).

Melting point analysis can be performed with PCRs utilising hybridisation probes and molecular beacons as they are not hydrolysed by Taq polymerases to generate signal. This step occurs immediately after the PCR assay. Melting curve analysis confirms the PCR product as the correct amplification product and can distinguish base pair differences (mutations or polymorphisms in target DNA) (9) e.g. genotyping Herpes simplex virus types 1 and 2 (19) and distinguishing multiple rifampin resistance mutations and high-level ioniazid resistance mutations in Mycobacterium tuberculosis (6).

Analysis is performed by raising the temperature while continuously monitoring the fluorescent signal. SYBR Green melting curve analysis allows monitoring the melting behaviour of the PCR products and allows discrimination between specific product and primer dimers (nonspecific binding to form non-specific products). Hybridisation probe melting curves detect the separation of target hybridisation probes. Probes that are bound to perfectly matching target DNA dissociate at a higher [T.sub.m] than DNA that contains sequence variation.

Molecular methods--other than PCR[TM] amplification These methods are based on signal, target, probe or rolling circle amplification.

Signal amplification techniques

The principle of signal amplification is to directly detect nucleic acids without target amplification by increasing the signalling capacity of the labelled molecules attached to the target nucleic acid eg. branched DNA probes (bDNA), Hybrid capture (anti-DNA-RNA hybrid antibody). Advantages of signal amplification methods over target amplification methods are that the system does not require a thermal cycler and the number of target molecules is not altered so the signal generated is directly proportional to the amount of target sequence present in the clinical specimen. These methods can be carried out at a single temperature (isothermal), are not affected by the presence of enzyme inhibitors, so simpler nucleic acid extraction methods can be used and are less susceptible to contamination. The method can directly measure RNA levels thus eliminating the requirement for cDNA synthesis (1).

bDNA probe methods utilise a branched multiple probe-enzyme complex. The primary probe captures the nucleic acid target onto a solid surface. Branched probes specific for the primary probes are added followed by enzyme-labelled probes which are detected by chemiluminescent substrates to produce a signal which can be quantitated.

An example of Hybrid Capture is the Digene HPV test. The HC2 High-Risk HPV DNA test uses Hybrid Capture 2 technology, a nucleic acid hybridisation assay utilising microplate chemiluminescent detection. Specific HPV RNA probes hybridise to target DNA. The resultant RNA: DNA hybrids are captured onto a microwell solid surface which is coated with antibodies specific for the hybrids. Alkaline phosphatase conjugated specific antibodies bind to the hybrids, and are detected by a chemiluminescent substrate. Multiple conjugated antibodies bind to each capture hybrid resulting in substantial signal amplification (22).

Target amplification systems (other than PCR)

These methods are similar to the PCR process as they use enzyme mediated processes and oligonucleotide primers that bind to complementary sequences on opposite strands of double stranded targets to synthesise copies of target nucleic acid (1). Detection is by hybridization probes, molecular beacons or chemiluminescent probes.

Transcription amplification methods

Isothermal based nucleic acid amplification methods e.g. nucleic acid sequence-based amplification (NASBA) and transcription-mediated amplification (TMA). These methods do not require a thermal cycler, have rapid kinetics, produce a result in a very short time frame and can be done in a single tube. The disadvantages are poor performance with DNA targets and the stability of complex multi-enzyme systems has been questioned (1).

An example of NASBA is the automated NucliSENS EasyQ system (BioMerieux). This system combines two technologies of amplification and detection. In an initial enzymatic amplification of nucleic acid target, single-stranded RNAs are generated. Detection occurs with exponential amplification of the target sequence when specific molecular beacon probes hybridize causing fluorescence (23). NASBA is able to specifically amplify and directly detect RNA target in a DNA background and can detect RNA without DNA contamination. RNA amplicons are single stranded and therefore do not require denaturation prior to detection.

An example of a TMA based assay is the Gen-Probe kit for the detection of Chlamydia trachomatis. The method involves a promoter primer that binds to the rRNA target. Reverse transcription creates cDNA. A second primer binds to cDNA and reverse transcription creates a new DNA copy. RNA is transcribed from the DNA template to produce many copies of target.

Strand displacement amplification (SDA)

Isothermal nucleic acid amplification of a particular sequence by a two step process--target generation and exponential target amplification. The primer containing a restriction site binds to the target and DNA is synthesised. The restriction enzyme nicks the strand containing the restriction site and polymerase begins amplification again, displacing the newly synthesised strand. This method has the advantage of being technically simple to perform (1).

An example of SDA based assay is the BD ProbeTec[TM] assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoea using simultaneous amplification of nucleic acids by SDA and real-time detection by using fluorescence resonance energy transfer (FRET) in a one hour format.

Probe amplification techniques

Probe amplification methods amplify products containing only the probe sequence. Some examples are Ligase Chain Reaction (LCR), Cleavase-Invader assays and Cycling Probe technology.

Ligase chain reaction (LCR)

The LCR differs from PCR because it ligates the probe molecule. Two probes are used for each DNA strand and are joined (ligated) together to form a single probe. Amplification is by thermal cycler and, since both the ligated product and the original strand can act as a template there is an exponential increase in product. To improve specificity, LCR commonly uses two enzymes, both a DNA polymerase enzyme and a DNA ligase enzyme, to drive the reaction so LCR can have greater specificity than PCR. For this gapped LCR (G-LCR), a short gap is left between the two annealed probes and is filled by the DNA polymerase before the remaining "nick" is filled (ligated) by DNA ligase, amplification will only occur if the probe has been ligated (1). An example of LCR--probe amplification, was the Abbott detection kit for C. trachomatis and N. gonorrhoea, but it is no longer commercially available.

Cleavase-invader technology

Invader assays (Third Wave Technology), invader probe amplification (IPA), (www.twt.com/invader_chemistry/invaderchem.htm) is a probe amplification system that detects nucleic acid target using specific probes, one of which overlaps. The overlapping probe region (flap) is cleaved by a DNA polymerase called Cleavase. The resulting cleaved molecule is amplified and serves as a template for a second reaction. The flap binds to a fluorescent labelled probe releasing fluorescence (FRET). Repeating the process amplifies the signal.

Cycling probe technology

Cycling probe technology (CPT; ID Biomedical Corp., Bothell, Washington) (http://bio.takara.co.jp/bio_en/catalog_d.asp?c_ID=C1265) employs a DNA-RNA-DNA fluorescent labelled probe at a constant temperature. The probe labelled with fluorescence one end and a Quencher the other, anneals to target DNA. An enzyme cuts the RNA region of the probe, the probe is no longer intact unquenching the signal, resulting in emission of fluorescence. Probe amplification is linear and not exponential. This application has been used to detect the mecA gene of MRSA. This method can be used for quantitative (1).

Rolling circle amplification (RCA)

Isothermal amplification, distinct from other isothermal signal, target or probe amplification. A DNA probe anneals to target DNA, the probe acts as a primer for RCA. The free end of the probe anneals to a small circular DNA template. DNA polymerase extends the primer generating linear strands of DNA consisting of multiple repeated copies that can be readily detected. Advantages are that the method is simple, robust, has good sensitivity and is able to be multiplexed.

Automated robotic molecular platforms

Automation of the molecular microbiology laboratory has the potential to assist streamlining of testing processes by offering reliability and standardisation, particularly in high-throughput laboratories. This is accomplished by removing the possibility of operator error and variation. Laboratory personal are also better protected from overuse syndrome due to performance of an excessive number of manual manipulations. Automated equipment can be purchased to perform one part of the PCR process, several or all of the steps required to obtain a result. Some automation systems only operate with pre-packaged applications.

Automated nucleic acid extractors

There is a wide range of DNA and/or RNA extractors available from various companies, capable of handling as few as six and up to several hundred samples per day. Samples that can be processed range from whole blood, serum, plasma, tissue, urine to aspirates such as CSF and sputum. Some extractors are designed to only process limited sample types. Some examples of nucleic acid extractors are listed in Table 1.

Automated PCR setup

Separate machines can also be purchased to prepare and set up PCR reactions.

Full system from extraction, amplification to detection

Complete performance of the molecular detection of microbiology pathogens can be performed by several automated systems. All that is required is the loading of the samples by laboratory personnel. Some examples are listed in Table 2.

Automated amplification and detection

There is a wide range of automated PCR amplification and detection systems available. However, some systems can only detect pathogens using the appropriate commercial kit. Some examples of automated amplification and detection systems are listed in Table 3.

A comparison of some of these systems can be viewed at

http://www.biocompare.com/matrix/2838/ Real-Time-PCR-ThermalCyclers--Thermocyclers.html

Emerging DNA-based detection technologies TM BioScience multiplex PCR--Luminex Technology

Luminex Technology utilises microspheres which are tiny color-codes beads that are divided into 100 distinct sets. Each bead set can be coated with a reagent specific to a particular bioassay of interest e.g. an oligonucleotide probe, allowing the capture and detection of specific microbiological pathogens from a sample. Within the Luminex analyzer, lasers excite the internal dyes that identify each microsphere bead, as well as any reporter dye captured during the assay. Many readings are made on each bead set, further validating the results. In this way, Luminex technology allows multiplexing of up to 100 unique assays within a single sample rapidly with sensitivity and specificity (24).

What next?

Lightcycler[R] SeptiFast test

This test is currently available for the detection and identification of bacterial and fungal DNA. Using a 3 ml whole blood sample, that does not require prior incubation or culture steps and producing a result in less than 6 hours. The LightCycler[R] SeptiFast Kit detects and identifies the 25 most important pathogens known to cause blood stream infections (25).

SmartSense[TM] is a bio-electronic technology that has been developed by BioWarn, LLC, which allows the real-time detection and identification of pathogens. The SmartSense[TM] sensor is based on a semiconductor chip to which a ligand (antibody) has been bound. When the ligand combines with its target (antigen), an electrochemical signal is sensed by the chip, amplified and processed to obtain pathogen identification. This technology enables low-cost, hand-held, highly sensitive robust detection capability. Since the system is semiconductor chip-based, the detector system can be reduced to very small size and low unit cost. SmartSense[TM] prototypes have been fabricated, laboratory tested, and have potential applications for H5N1 virus, MRSA, HIV, opportunistic respiratory infections and VRE (26,27).

MicroArray technology may use a PCR amplified product. This step minimises the amount of specimen required from a clinical sample as well as increasing the sensitivity of the assay. This product binds specifically (hybridises) to probes of interest present on a DNA hybridisation chip. Many probes can be present on the microchip enabling multiple screening in the one test. This type of testing is particularly useful for emerging pathogens requiring multiplexing assays where the typing and sub-typing of the organism can be obtained in the one test. This application has the potential to be miniaturised, enabling bedside testing (hand-held gene analysers), and thus will alter the future implementation and application of DNA diagnostic assays. Currently the cost of this methodology limits its use in the laboratory.

Bio-defense detection

With the emergence of bio-terrorism rapid, hands- free, closed systems (to avoid contamination or carryover) that can be used in the field are an important area that is being developed for the detection of organisms such as anthrax, plague, smallpox, ricin toxin, Staphylococcal enterotoxin-B and avian flu (27). Molecular detection is also required for possible agents of bio-terrorism to provide rapid discrimination of weaponised pathogens from harmless laboratory-adapted or vaccine-related strains (13). An example is the use of Real time PCR to provide rapid screening for the presence of Bacillus anthracis spores (6).

The molecular world is constantly developing and has huge potential. Current methods can be improved to decrease detection time and further enhance specificity and sensitivity. As PCR cannot satisfy all the molecular needs of the microbiology laboratory, non-PCR amplification methods are being developed further. Molecular detection is a powerful tool for the detection of infectious agents in the medical microbiology laboratory and advances in this field will greatly enhance patient care.

Molecular testing technologies currently available in New Zealand can be viewed in Table 4.

[FIGURE 1 OMITTED]

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[FIGURE 3 OMITTED]

Acknowledgement

I wish to thank John Mackay, molecular biologist, Linneaus Limited, Gisborne for his expert assistance in the development of this manuscript.

References

(1.) Nolte FS, Caliendo AM.. Manual of Clinical Microbiology (8th ed). Murray et al., (eds). ASM Press, USA. 2003. Ch 17, pp 234-256.

(2.) Roche. Polymerase Chain Reaction. Roche Diagnostics GmbH, Germany. 1998.

(3.) Roche.Roche Molecular Biochemicals PCR Applications Manual. 2nd edition. Roche Diagnostics GmbH, Germany. 1999.

(4.) Roche. The Evolution of PCR. Roche Diagnostics GmbH, Germany. 2003.

(5.) Becker K. Principles and role of nucleic acid amplification and modern microbiological diagnosis (Article in German). Anasthesiol Intensivemed Notfallmed Schmerzther 2000; 35: 35-40.

(6.) Kaltenboeck B, Wang C. Advances in real-time PCR: application to clinical laboratory diagnostics. Adv Clin Chem 2005; 40: 219-59.

(7.) Richman DD, Cleveland PH, Redfield DC, Oxman MN, Wahl GM. Rapid viral diagnosis. J Infect Dis 1984; 149: 298-310.

(8.) Brunk CF, Avaniss-Aghajani E, Brunk CA. A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences. Appl Environ Microbiol 1996; 62: 872-9.

(9.) Cockerill FR 3rd. Application of rapid-cycle real-time polymerase chain reaction for diagnostic testing in the clinical microbiology laboratory. Arch Pathol Lab Med 2003; 127: 1112-20.

(10.) Mackay IM. Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 2004; 10: 190-212.

(11.) Syrmis MW, Whiley DM, Thomas M, Mackay IM, Williamson J, Siebert DJ, et al. A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples. J Mol Diagn 2004; 6: 125-31.

(12.) Al-Soud WA, Radstrom P. Purification and characterisation of PCR-inhibitory components in blood cells. J Clin Microbiol 2001; 39: 485-93.

(13.) Normandin M, Tsongalis GJ. Detection methods for nucleic acid amplification products. In: Laboratory Diagnosis of Viral Infections. EH Lennette, TF Smith (Eds.). Marcel Dekker, Inc, New York. USA. 1999. Ch 8.

(14.) http://www.cambrex.com/Content/bioscience/CatNav.asp?oid=728 &prodoid=NusieveGTG

(15.) Molecular Probes. SYBER Safe[TM] DNA Gel stain. Molecular Probes, Inc. USA. 2004.

(16.) Sanger F, Nicklen S, Coulsen AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977; 74: 5463-7.

(17.) Mackay J, Landt O. Real-time PCR fluorescent chemistries. Methods Mol Biol 2007; 353: 237-61.

(18.) Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006; 19: 165-256.

(19.) Evans GE, Beynon KA, Chua A, Anderson TP Werno A, Jennings LC, et al. Comparison of real-time PCR with culture and EIA for the diagnosis of mucocutaneous infection with herpes simplex virus. N Z J Med Lab Sci 2006; 60: 92-6.

(20.) http://dorakmt.tripod.com/genetics/glosrt.html

(21.) http://www.allelogic.com

(22.) http://www.digene.com/labs/labs_hpv_01.html

(23.) http://www.biomerieux-usa.com/clinical/nucleicacid/ easyq/easyq_technology.htm

(24.) http://www.luminexcorp.com/technology/index.html

(25.) http://www.roche.com/prod_diag_lc-septif.html

(26.) Riggs JL. New developments in identification of microorganisms and chemicals. 9th International Conference, Detection Technologies, 2006, San Diego, California, USA.

(27.) http://www.biowarnllc.com

Gloria Evans, Dip MLS, MMLSc (Dist), FNZIMLS

Laboratory for Cell and Protein Regulation, Department of Obstetrics and Gynaecology, University of Otago,

Christchurch

Address for correspondence: Gloria Evans. Laboratory for Cell and Protein Regulation, Department of Obstetrics and Gynaecology, University of Otago, Christchurch. Email: gloria.evans@otago.ac.nz
Table 1. Automated nucleic acid extractors.

Nucleic acid extractor systems   Manufacturer

ABI PRISM[TM] 6100               Applied BioSystems
Biomek[R][FX.sup.P] including    Beckman Coulter
  PCR setup
NucliSENS[R] easyMAG[TM]         Biomerieux

X-Tractor Gene[TM]               Corbett Life Science
AutoGenPrep                      GenPrep
Iprep[R] purification            Invitrogen
Magtration Systems               Precision System Science
Maxwell[R]16 System              Promega
QIAcube BioRobot MDx MDx DSP     Qiagen
  workstation
COBAS[R] Ampliprep[R] System     Roche
  MagNa Pure LC Instrument

Nucleic acid extractor systems   Website

ABI PRISM[TM] 6100               www.appliedbiosystems.com/
Biomek[R][FX.sup.P] including    www.beckmancoulter.com/products/
  PCR setup                        instrument/automatedsolutions/
NucliSENS[R] easyMAG[TM]         www.biomerieux-usa.com/clinical/
                                   nucleicacid/easymag/index.htm
X-Tractor Gene[TM]               www.corbettlifescience.com
AutoGenPrep                      www.autogen.com/Products/
Iprep[R] purification            https://catalog.invitrogen.com/
Magtration Systems               www.pss.co.jp/english/products/
                                   02.html
Maxwell[R]16 System              www.promega.com/maxwell16/default.htm
QIAcube BioRobot MDx MDx DSP     www1.qiagen.com/Products/Automation/
  workstation
COBAS[R] Ampliprep[R] System     http://us.labsystems.roche.com/
  MagNa Pure LC Instrument         products/molecular/

Table 2. Complete automated molecular detection systems.

Complete molecular systems              Manufacturer

Utilises a mechanised nucleic acid      Roche
  extractor which is teamed with a
  separate amplification/detection
  system, such as the
--COBAS[R] Ampliprep[R]/COBAS[R]
    Amplicor[TM] for PCR,
--COBAS[R] AmpliPrep[R]/COBAS[R]
    TagMan[TM] System, real-time PCR
    of up to four simultaneous
    assays.
BioRobot Universal.                     Qiagen
Automated RT PCR, PCR, sequencing
  reaction, gene expression
  analysis, genotyping and forensic
  assays.

Complete molecular systems              Website

Utilises a mechanised nucleic acid      http://www.roche.com/
  extractor which is teamed with a        prod_diag_pcr.htm
  separate amplification/detection
  system, such as the
--COBAS[R] Ampliprep[R]/COBAS[R]
    Amplicor[TM] for PCR,
--COBAS[R] AmpliPrep[R]/COBAS[R]
    TagMan[TM] System, real-time PCR
    of up to four simultaneous
    assays.
BioRobot Universal.                     www1.qiagen.com/
Automated RT PCR, PCR, sequencing         products/automation
  reaction, gene expression
  analysis, genotyping and forensic
  assays.

Table 3. Automated amplification and detection

Amplification and detection system        Manufacturer

GeneAmp7300RT, Prism 7500                 Applied Biosystems
NucleSENS[R] (NASBA)                      Biomerieux
DNA Engine Opticon                        BioRad
SmartCycler[R]                            Cepheid
Rotor-GeneTM 6000 RT cycler               Corbett Life Science
Mastercycler[R] ep realplex               Eppendorf
Rapidcycler[R] 2                          Idaho Technology
Cobas[R] Amplicor[R] Analyzer             Roche
  (PCR), commercial kits.

Real Time Detection
Lightcycler[R] 2.0
LightCycler 480 real-time PCR System
Cobas[R] Taqman[R] 48 analyzer for
  detection of HIV, HBV, HCV nucleic
  acid and as many as 10 different
  user-defined PCR profiles
Cobas[R] Taqman[R] 96 analyzer
  automated, for the amplification
  and detection of viruses like HIV,
  HBV, and HCV.
Mx300P[TM]                                Stratagene

Amplification and detection system        Website

GeneAmp7300RT, Prism 7500                 https://products.
                                            appliedbiosystems.com/
NucleSENS[R] (NASBA)                      www.biomerieux-usa.com
DNA Engine Opticon                        www.biorad.com
SmartCycler[R]                            www.cepheid.com
Rotor-GeneTM 6000 RT cycler               www.corbettlifescince.com
Mastercycler[R] ep realplex               www.eppendorf.com
Rapidcycler[R] 2                          www.idahotech.com/
                                            rapidcycler/
Cobas[R] Amplicor[R] Analyzer             www.roche.com/
  (PCR), commercial kits.                   prod_diag-pcr.htm

Real Time Detection
Lightcycler[R] 2.0
LightCycler 480 real-time PCR System
Cobas[R] Taqman[R] 48 analyzer for
  detection of HIV, HBV, HCV nucleic
  acid and as many as 10 different
  user-defined PCR profiles
Cobas[R] Taqman[R] 96 analyzer
  automated, for the amplification
  and detection of viruses like HIV,
  HBV, and HCV.
Mx300P[TM]                                www.stratagene.com

                         Testing
                         laboratory and
                         method of detection
Pathogen                 (where available)

Adenovirus               A+CHL (2,8)

Aspergillus spp.         CHL (2)

Atypical
Mycobacterium spp.
(refer Mycobacterium
tuberculosis)

Avian Influenza          A+ (1)
                         ESR (3)
                         CCHL
                         CHL (3)

Bartonella henselae      A+ (8)
(Cat Scratch Disease)

BK virus                 A+CHL

Bordetella               A+,
pertussis                Waikato
                         CCHL (1)
                         CHL (3)

Chlamycha                A+ (5)
trachomatis              Waikato (6)
                         CCHL (4)
                         APATH (6)
                         CHL (5)
                         Various other
                         laboratories

CJD                      Australian CJD
                         registry, Melbourne
                         University

Coronavirus (refer
human coronavirus)

Coxsackie virus
(refer Enterovirus)

Cytomegalovirus (CMV)    A+,
                         CCHL
                         CHL1,Z

Echovirus (refer
Enterovirus)

Enterovirus--Coxsackie   A+
A and B, Echovirus,      Waikato (1)
Enterovirua,             CCHL
Poliovirus               ESR
                         CHLZ

Epstein Barr virus       A+1
(EBV)

Hepatitis B (HBV)        A+
                         ESR (6)

Hepatitis C (HCV)        A+ (4)
                         CCHL (4) (qual.
                         only) ESR
                         CHL (1) (qual.&
                         genotyping only)

Hepatitis D (HDV)        A+ (6)

Herpes simplex virus     A+
(HSV 1 and 2)            Waikato (1)
                         CCHL (1)]
                         APATH (1)
                         CHL (1,2)

Herpes zoster virus
(refer varicella
zoster)

Human coronavirus        CHL (3,8)

Human herpes virus       A+
type 6 (HHV6)            CHL1

Human herpes virus       A+
type 8 (HHV8)

HIV                      A+
                         Waikato
                         CCHL (4)
                         CHL (4) (HIV VL
                         only)

HIV drug resistance      A+
genotying

Human papilloma          A+
virus (HPV)              Waikato (7)

Influenza A, B           A+ (1)
                         ESR (3)
                         CHL (3)

JC virus                 A+
                         CHL

Legionella spp.          Waikato
                         ESR
                         CHL (2,8)

Leptospira spp.          ESR
                         CHL (3)

Measles virus            A+
                         CHL (3)

Meningococcal
PCR--refer Neisseria
meningitis PCR

Metapneumovirus          CHL (1,8)

Mycobacterium-           A+ (8)
tuberculosis (TB)        CHL (4)

Mycoplasma               APATH (1)
genitalium

Mycoplasma               CHL (2)
pneumoniae

Neisseria                A+
meningitidis             Waikato
                         CCHL (1)
                         ESR
                         CHL (1)

Norovirus                CHL (2)
                         ESR (2) (typing)

Papilloma virus
(HPV) refer human
papilloma virus

Parainfluenza            CHL (3,8)
viruses 1,2,3

Parvovirus B19           A+
                         CHL (3)

Picornavirus             CHL (3,8)
(Enterovirus and
Rhinovirus)

Polio (refer
Enterovirus)

Respiratory Syncytial    CHL (3,8)
Virus

Rhinovirus (refer
Picornavirus)

Rickettsia spp.          A+ (8)

Rubella                  A+ (1)

SARS (coronavirus)       A+
                         CCHL (1)
                         CHL (1,3,8)

Streptococcus            ESR
pneumoniae               CCHL

Toxoplasma gondii        A+
                         CHL (1)

Trichomonas              APATH (1)
vaginalis

Tropheryma whipplei      A+

Ureaplasma parvum        APATH (1)

Ureaplasma               APATH (1)
urealyticum

Varicella zoster         A+
(VZ)                     Waikato
                         CCHL
                         CHL (1,2)

Whipple's disease
(refer Tropheryma
whipplei

Additionally             A+ (8)
Bacterial species

Pathogen                 Sample type

Adenovirus               Respiratory--nasopharyngeal
                         swab or aspirate, sputum.
                         Urine (A+, CCHL). Faeces,
                         tissue biopsy (A+).

Aspergillus spp.         EDTA blood.

Atypical
Mycobacterium spp.
(refer Mycobacterium
tuberculosis)

Avian Influenza          Respiratory--nasopharyngeal
                         aspirate (NPA), pernasal swab,
                         sputum.

Bartonella henselae      EDTA or CPD blood, tissue.
(Cat Scratch Disease)

BK virus                 Urine and EDTA blood,
                         cerebospinal fluid (CSF).

Bordetella               Dry nasopharyngeal swab.
pertussis

Chlamycha                First catch urine (FCU),
trachomatis              endocervix/urethral swabs. Eye
                         swab (CCHL).

CJD                      CSF 0.5 ml, 20 mL EDTA whole
                         blood.

Coronavirus (refer
human coronavirus)

Coxsackie virus
(refer Enterovirus)

Cytomegalovirus (CMV)    EDTA blood, tissue, CSF,
                         bronchoalveolar lavage (BAL),
                         urine, amniotic fluid,
                         respiratory.

Echovirus (refer
Enterovirus)

Enterovirus--Coxsackie   CSF. Acute phase serum (A+).
A and B, Echovirus,
Enterovirua,
Poliovirus

Epstein Barr virus       CSF, fresh tissue
(EBV)                    [A.sup.+].EDTA blood,
                         unseparated, stored at room
                         temperature, CCHL.

Hepatitis B (HBV)        Serum, liver biopsy.

Hepatitis C (HCV)        Serum, liver biopsy.

Hepatitis D (HDV)        Serum, liver biopsy.

Herpes simplex virus     CSF, tissue, lesion swab,
(HSV 1 and 2)            BAL.

Herpes zoster virus
(refer varicella
zoster)

Human coronavirus        Respiratory--nasopharyngeal
                         swab (NPS), lung, sputum.

Human herpes virus       EDTA blood, CSF.
type 6 (HHV6)

Human herpes virus       EDTA blood, serum, biopsy.
type 8 (HHV8)            Karposi sarcoma, bone marrow
                         (BM).

HIV                      Qualitative 1 x EDTA blood.
                         Quantitative 2 x EDTA blood.

HIV drug resistance      1 x EDTA blood.
genotying

Human papilloma          Tissue, biopsy, genital swab,
virus (HPV)              scraping, cytobrush.

Influenza A, B           Respiratory--NPA, pernasal,
                         sputum.

JC virus                 CSF, urine.

Legionella spp.          Serum, urine, sputum, BAL.

Leptospira spp.          Acute--CSF, serum/EDTA plasma.
                         Convalescent-urine, tissue.

Measles virus            CSF, EDTA blood/serum, fresh
                         brain biopsy, NPS.

Meningococcal
PCR--refer Neisseria
meningitis PCR

Metapneumovirus          Respiratory--NPS, sputum.

Mycobacterium-           CSF, tissue, pleural or
tuberculosis (TB)        pericardial fluid, bone
                         marrow, fine needle aspirate
                         (FNA). Paraffin embedded
                         tissue (A+).

Mycoplasma               Genital swab.
genitalium

Mycoplasma               Throat swab, sputum, BAL.
pneumoniae

Neisseria                CSF, EDTA blood.
meningitidis             Fresh tissue (CCHL).

Norovirus                Faeces, vomit (sometimes if
                         no faeces available).

Papilloma virus
(HPV) refer human
papilloma virus

Parainfluenza            Respiratory--NPS, BAL.
viruses 1,2,3

Parvovirus B19           Serum, bone marrow in EDTA,
                         throat swab (TS), amniotic
                         fluid, foetal blood, post
                         mortem tissue, CSF, joint
                         aspirate.

Picornavirus             Respiratory--NPS, sputum.
(Enterovirus and
Rhinovirus)

Polio (refer
Enterovirus)

Respiratory Syncytial    Respiratory--NPS, sputum.
Virus

Rhinovirus (refer
Picornavirus)

Rickettsia spp.          EDTA blood

Rubella                  CSF, EDTA blood, fresh
                         placental biopsy, fetal blood.

SARS (coronavirus)       Respiratory--NPS, sputum,
                         lung. Faeces, serum/EDTA
                         plasma, CSF.

Streptococcus            CSF, PM samples. EDTA blood
pneumoniae               (CCHL, ESR) Tissue aspirates
                         (ESR).

Toxoplasma gondii        Acute--CSF, EDTA blood,
                         tissue. Intrauterine
                         infection--amniotic fluid
                         (AF), fetal blood (EDTA).
                         Congenital infection--AF,
                         placental biopsy, cord blood
                         (EDTA).

Trichomonas              Genital swab.
vaginalis

Tropheryma whipplei      CSF, fresh tissue.

Ureaplasma parvum        Genital swab.

Ureaplasma               Genital swab.
urealyticum

Varicella zoster         CSF, EDTA blood, (A+, CCHL),
(VZ)                     tissue, skin/genital swab.

Whipple's disease
(refer Tropheryma
whipplei

Additionally             Any sample where organisms
Bacterial species        are seen on Gram stain.

Pathogen                 Notes/comments

Adenovirus               Alternative-cell culture,
                         method of choice (CHL). Red
                         eye, pneumonia, hepatitis,
                         encephalitis, haemorrhagic
                         cystitis, gastroenteritis.

Aspergillus spp.         Alternative--culture, slow
                         growing and requiring
                         specialised culture
                         media. Fungal PCR. Assay is
                         specific for pathogenic
                         Aspergillus spp.

Atypical
Mycobacterium spp.
(refer Mycobacterium
tuberculosis)

Avian Influenza          Bird flu. Not cultured due to
                         highly infectious nature of
                         specimen. H5N1 only (CCHL).

Bartonella henselae      Alternative--culture, slow
(Cat Scratch Disease)    growing and requiring
                         specialised culture media;
                         formerly named Rochalimea
                         henselae.

BK virus                 Polyoma virus. Haemorrhagic
                         cystitis.

Bordetella               Alternative--culture, slow
pertussis                growing and requiring
                         specialised culture media.
                         Pertussis, whooping cough. PCR
                         remains positive longer than
                         culture. Confirmation and
                         typing (ESR).

Chlamycha                Alternative--cell culture,
trachomatis              slow growing and requiring
                         specialised cell culture.
                         Lymphogranuloma venereum (LGV)
                         typing (APATH).

CJD                      Specimens forwarded from CHL.
                         Creutzfeldt-Jakob disease, 14-
                         3-3 protein, prion protein
                         gene, PRNP, spongiform
                         encephalopathy. CJD
                         polymorphism risk factor codon
                         129.

Coronavirus (refer       SARS, human coronavirus.
human coronavirus)       Common cold.

Coxsackie virus          Hand foot and mouth disease.
(refer Enterovirus)

Cytomegalovirus (CMV)    Alternative--cell culture of
                         tissue and urine, slow growing
                         virus. Qualitative and
                         quantitative PCR. CMV drug
Echovirus (refer         resistance testing, EDTA blood
Enterovirus)             (A+).

Enterovirus--Coxsackie   Alternative//cell culture,
A and B, Echovirus,      slow growing virus, samples
Enterovirua,             sites vesicle fluid, throat,
Poliovirus               and rectum. Culture enables
                         typing of virus. CSF culture
                         not as successful as PCR
                         detection (low viral load).
                         Polio typing PCR/ELISA from
                         culture isolate (ESR).

Epstein Barr virus       Infectious mononucleosis,
(EBV)                    glandular fever. Quantitative
                         PCR.

Hepatitis B (HBV)        Quantitative. HBV YMDD
                         mutation, serum (A+).

Hepatitis C (HCV)        HCV qualitative, quantitative
                         and genotyping,

Hepatitis D (HDV)        HDV

Herpes simplex virus     Alternative cell culture, DFA
(HSV 1 and 2)            (where indicated) Typing by
                         Real time PCR using meltback
                         analysis for virus typing. In
                         house PCR, typing by
                         restriction endonuclease (RE)
                         digestion for CSF (CHL)

Herpes zoster virus      HZV
(refer varicella
zoster)

Human coronavirus        (not SARS) HCV 229E, HCVOC43

Human herpes virus       Roseola infantum, xanthema
type 6 (HHV6)            subitum, Sixth disease.

Human herpes virus       Considered a necessary
type 8 (HHV8)            prerequisite for Karposi's
                         sarcoma. Association with a
                         number of conditions including
                         multicentric Castleman's
                         Disease, multiple myeloma and
                         in primary effusion lymphomas
                         (A+).

HIV                      Human immunodeficiency virus,
                         AIDS, retroviral illness. HIV
                         viral load (VL). Qualitative,
                         quantitative PCR. Pro-viral
                         HIV DNA (HIV1 and HIV2) (A+).
                         HIV genotype resistance
                         testing (A+).

HIV drug resistance      HIV resistance testing.
genotying

Human papilloma          HPV cannot be cultured.
virus (HPV)              Genital warts, papilloma virus

Influenza A, B           Alternative, DFA, Rapid
                         testing, cell culture.A+
                         influenza A only. Refer Avian
                         flu separately.

JC virus                 Polyoma virus. Jamestown
                         Canyon virus.

Legionella spp.          Alternative culture, slow
                         growing fastidious bacteria,
                         requiring specialised media,
                         recovery rate low. Sequencing
                         for speciation (ESR, CHL).

Leptospira spp.          Alternative culture, slow
                         growing spirochaete and
                         requiring specialised media.

Measles virus            Morbillivirus, English measles.

Meningococcal
PCR--refer Neisseria
meningitis PCR

Metapneumovirus          Non culturable. Non routine.

Mycobacterium-           Alternative culture--slow
tuberculosis (TB)        growing bacteria and requiring
                         specialised media. M.
                         tuberculosis, M. bovis, M.
                         bovis (BCG). HSP65 for
                         Mycobacterium spp. M.
                         tuberculosis, M. avium-
                         complex and M. gordonae by
                         Rapid DNA probes (A+), M.
                         tuberculosis, M. avium and M.
                         intracellularae (CHL).

Mycoplasma               Multiplex PCR with T.
genitalium               vaginalis, U. urealyticum and
                         U. parvum also detected.

Mycoplasma               Alternative culture--slow
pneumoniae               growing bacteria and requiring
                         specialised media.

Neisseria                Alternative bacterial culture.
meningitidis             Meningococcal meningitis.
                         Characterisation by sequencing
                         (ESR).

Norovirus                Nonculturable. Calicivirus,
                         Norwalk-like virus (NLV),
                         small round-structured virus
                         (SRSV). Genotyping by DNA
                         sequencing (ESR).

Papilloma virus
(HPV) refer human
papilloma virus

Parainfluenza            Alternative cell culture.
viruses 1,2,3

Parvovirus B19           Erythrovirus B19. Erythema
                         infectiosum (fifth disease,
                         "slapped cheek syndrome").

Picornavirus             Alternative cell culture, slow
(Enterovirus and         growing virus.
Rhinovirus)

Polio (refer             Acute flaccid paralysis, AFP.
Enterovirus)

Respiratory Syncytial    Alternative cell culture. RSV
Virus

Rhinovirus (refer        Common cold.
Picornavirus)

Rickettsia spp.          Typhus Fever group
                         Spotted Fever group
                         Tsutsugamushi Fever group
                         Culture difficult and
                         potentially dangerous.

Rubella                  German measles.

SARS (coronavirus)       Severe Acute Respiratory
                         Syndrome. SARS-Cov, Urbani
                         virus, HcoV

Streptococcus
pneumoniae

Toxoplasma gondii        Toxoplasmosis, protozoan
                         parasite.

Trichomonas              Trich. Multiplex PCR with M.
vaginalis                genitalium, U.urealyticum and
                         U. parvum also detected.

Tropheryma whipplei      Whipple's disease. T.
                         whippelei. Tropheryma whipplei
                         culture difficult and
                         insensitive.

Ureaplasma parvum        Multiplex PCR with M.
                         genitalium, T. vaginalis and
                         U.urealyticum also detected.

Ureaplasma               Multiplex PCR with M.
urealyticum              genitalium, T. vaginalis and
                         U. parvum also detected.

Varicella zoster         Alternative--culture,
(VZ)                     difficult to grow. Chicken
                         pox, shingles, VZV, VZ, HZV,
                         herpes zoster, zoster. CSF In
                         house PCR (CHL).

Whipple's disease
(refer Tropheryma
whipplei

Additionally             Universal primers for 16S rRNA
Bacterial species        amplification and sequencing.
                         For speciation to genus level
                         (and sometimes to species
                         level). The test is for
                         guidance only and in
                         conjunction with clinical
                         consultation.

Molecular detection method:

1. Real time PCR
2. PCR in house
3. PCR in house nested
4. Cobas Amplicor (PCR and ELISA)
5. SDA - Strand displacement amplification
6. Real time PCR (Cobas Taqman)
7. Nucleic acid hybridisation with signal amplification (Digene)
8. Requires consultation with the Microbiologist

References for testing laboratories:

A+ - Labplus, Auckland City Hospital, Auckland. Laboratory Handbook

2007. www.labplus.co.nz/microbiology.htm.

ESR--Environmental Science & Research, Wellington. www.esr.cri.nz/
SearchResults.aspx?query=pcr

Waikato--Waikato Laboratories, Hamilton. www.waikatodhb.govt.nz/
laboratory/molecular_biology.htm.

CCHL--Capital Coast Health Laboratory Wellington Hospital, Wellington.
www.ccdhb.org.nz/HHS/lab/GenericList.asp.

APATH--Aotea Pathology, Wellington. Dr Collette Bromhead, personal
communication (0800 500430).

CHL--Canterbury Health Laboratories, Christchurch. www.cdhb.govt.
nz/chlabs/testsframe.htm.
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No portion of this article can be reproduced without the express written permission from the copyright holder.
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Author:Evans, Gloria
Publication:New Zealand Journal of Medical Laboratory Science
Geographic Code:8NEWZ
Date:Nov 1, 2007
Words:9047
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