Rapid and Simple Detection of Escherichia coli by Loop-Mediated Isothermal Amplification Assay in Urine Specimens.
Diagnosis and early detection of Urinary Tract Infection (UTI) are critical factors in disease management. Urine culture is the gold standard for the detection of UTI, but this method is time consuming. Molecular techniques are the more sensitive and rapid diagnostic tools for detecting pathogens in clinical samples. Conventional amplification methods such as PCR, require sufficient skill and expensive devices. In this regard, several isothermal amplification methods for the rapid detection of UTIs have been developed such as Loop mediated isothermal Amplification method (LAMP) (1), Nucleic Acid Sequence Based Amplification (NASBA) (2), Self-sustained sequence replication (3SR), etc. Compared to Conventional amplification methods, isothermal amplification techniques especially LAMP is rapid and easy to use for early detection of DNA. The thermocyclers are not needed in this amplification method, so procedure can be completed under isothermal conditions (1,2).
In this study, the performance of LAMP based on the mal B gene was assessed to detect the presence of Escherichia coli (E. coli) in urine specimens.
Materials and Methods
Samples collection and bacterial culture
Urine specimens collected from patients referring to Resalat Hospital in Iran. E. coli (ATCC 25922) was used to evaluate the specificity and sensitivity of the LAMP reaction and grown in LB broth medium.
(1.)5 ml of urine was poured into a microfuge tube. The tubes were centrifuged at 14000 rpm for 10 min. After washing with PBS buffer, samples were centrifuged again. Remaining Precipitate was solved in PBS buffer and incubated at 90 [degrees]C for 10 min then was subjected to reaction components of LAMP.
E. coli-specific Primers used for this study targeted malB gene with GenBank accession no. CP016358 which encodes Maltoprotein. A set of four specific primers (F3, B3 for outer primers and FIP, BIP for inner primers), as described by Hill et al (3), were employed for LAMP assay (Table l).
Final concentration of the LAMP reaction mix was prepared in total 25 [micro]l volume containing 0.2 Mm F3 and B3 primers, 1.6 [MICRO]M FIP and BIP primers, 1.4 mM dNTPs mixture, l mM betaine (Sigma, Shanghai, China), 6 mM MgS04, 1x LAMP buffer (New England Biolabs, Ipswich, MA), 320 U/m/Bst DNA polymerase (New England Biolabs). The reaction mixture was incubated at 66 [degrees]C for l hr using heating block (HB-R48, Wisdcopany, Korea).
Detection of amplification products
To detect LAMP products, electrophoresis was performed in 1% agarose gel (Figure 1). The LAMP reaction evaluation was also done by addition Syber green (1:10 dilution of a 10,000x stock solution) to each reaction tubes and color changes was visualized by UV lamp (302 nm). Green color indicates a positive result (Figure 1).
Specificity and sensitivity of LAMP
To determine the sensitivity of the assays, 10-fold serial dilutions purified DNA derived from E. coli ATCC 25922 (Table 2) were prepared. Extracted DNAs of several bacterial strains were examined to evaluate the specificity of the LAMP assay.
Microbiological and molecular Examinations of the urine samples
Macroscopic urinalysis was done by direct visual examination of the urine samples to which a small amount of acid was added. For microbiological tests (Table 3), all urine samples collected from patients were grown in LB broth medium. To determine whether there are E. coli, all of the urine samples were cultured on Eosin Methylene Blue Agar (Merck, Germany). The observation of green metallic sheen was considered positive. For LAMP assay (Table 3), 1 ml of each urine samples was centrifuged at 15000 rpm, A[degrees]C for 10 min. Then, the resultant precipitate was resus-pended in sterile water. Once incubated at 90[degrees]C for 10 min, the suspensions were added to LAMP reactions.
LAMP assay showed the best detection limit of DNA copies with 1.02 copies ([10.sup.-8] dilution) (Figure 2). In this study, DNA copy number of each dilution was calculated using the following formula (4):
DNA copy number =(6.022 x [10.sup.23])xC(ng)/650(1x[10.sup.9])xDNA lenght
In which C is concentration of the extracted DNA. DNA length is bases pair number of the selected bacterium which was 5130767 base pairs for E. coli ATCC 25922 in our study. No cross reaction was seen with other bacteria, indicating that designed primers of LAMP assay were specific for mal B gene (Figure 3).
As expected, LAMP was more specific and sensitive than direct microscopic tests. Among the 20 urine samples, 12 cases were positive on EMB agar, but 13 cases were positive by LAMP. We were not capable of detecting E. coli contaminated urine sample by culturing. The reason of this observation could be inability of bacteria to grow on culture medium due to antibiotics consumption by patients. Since DNA is stable for long time and it is detectable after bacteria death, so it would be expectable that DNA-based molecular detection techniques like LAMP could detect DNA in antibiotic-killed bacteria as well.
Diagnosis of UTI is often carried out by clinical symptoms, microscopic and culture examinations. Direct microscopic diagnosis has a high error rate (approximately 31%) and culture based methods are time consuming (5). The advantages of nucleic acid amplification based methods such as PCR, are their speed and sensitivity in pathogen detection, but these assays need trained users and some expensive devices (6). In this study, one of isothermal nucleic acid amplification methods called LAMP was employed to overcome the mentioned problems in UTIs diagnosis procedures.
LAMP could recognize target gene by 4-6 primers and the amplification can be done in constant temperature for 60 min. Unlike with PCR, there is no need for thermocycler and heat denaturation of the double strand DNA (7).
There are some similar researches in which LAMP assay has been targeted different genes to detect various strains of E. coli such as the stxl, stx2 (8), aggR (9), ipaH (10), the heat-labile 1 (LT1) and heat-stable I (ST1) genes " and mal B (3). Although many researches have been done to detect E. coli by LAMP method, but there is not any report for employing this technique to identify E. coli in urine samples. In spite of more than 90% similarity between Shigella and E. coli, no cross reaction was observed between them in this study. The reason of high specificity of LAMP method is recognition of six distinct sequences on the target DNA by a set of four specially designed primers .
In conclusion, the present study suggests that LAMP method would be highly suitable for the detection of the UTIs and also comfort quick diagnosis of UTI in clinical laboratories with limited equipment. However, LAMP assay is preferable to microbiological culture in terms of speed, simplicity and sensitivity.
The authors acknowledge the financial support of this investigation by Alzahra University (Tehran, Iran).
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Reihaneh Ramezani (1*), Zahra Kardoost Parizi (2), Nassim Ghorbanmehr (2), and Hamideh Mirshafiee (3)
1 Department of Biomedical Sciences, Women Research Center, Alzahra University, Tehran, Iran
(2.) Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
(3.) Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran
(*) Corresponding author: Reihaneh Ramezani, Ph.D., Department of Biomedical Sciences, Women Research Center, Alzahra University, Tehran, Iran
Tel: +98 21 85692095
Fax: +98 21 88049809
Received: 16 May 2017
Accepted: 28 Aug 2017
Table 1. Primers used in this study Assay Primer Sequence (5'- 3') F3 5'-GCCATCTCCTGATGACGC-3' B3 5'-ATTTACCGCAGCCAGACG-3' LAMP FIP 5'-CTGGGGCGAGGTCGTGGTAT- TCCGACAAACACCACGAATT-3' BIP 5'-CATTTTGCAGCTGTACGCTCGC- AGCCCATCATGAATGTTGCT-3' Tabie 2. Concentration and number of DNA copy for each sample Samples Dilution Number of DNA copy A [10.sup.-1] 1.02x[10.sup.7] B [10.sup.-2] 1.02x[10.sup.6] C [10.sup.-3] 1.02x[10.sup.5] D [10.sup.-4] 1.02x[IO.sup.4] E [10.sup.-5] 1.02x[10.sup.3] F [10.sup.-6] 1.02x[10.sup.2] G [10.sup.-7] 1.02x[10.sup.1] H [10.sup.-8] 1.02 Table 3. The results of all experiments (turbidity, culture, PCR and LAMP) for clinical specimens Samples Sex Age Turbidity Culture LAMP result (a) C1 F 35 + + + C2 F 45 + + + C3 F 63 + - - C4 F 12 + - - C5 M 36 + - - C6 F 42 + + + C7 M 43 + - - C8 F 56 + - - C9 F 37 + + + C10 F 59 - - - C11 F 63 + + + C12 M 60 + - + C13 F 57 + + + C14 M 33 + + + C15 F 40 + + + C16 F 64 + + + C17 M 39 + + + C18 M 66 + + + C19 F 27 + - - C20 M 35 + + + (a +), amplification occurred; -, amplification did not occur.
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|Title Annotation:||Short Communication|
|Author:||Ramezani, Reihaneh; Ghorbanmehr, Nassim; Mirshafiee, Hamideh|
|Publication:||Avicenna Journal of Medical Biotechnology (AJMB)|
|Date:||Oct 1, 2018|
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