Original Article

A Novel and Rapid Isothermal Nucleic Acid Based Detection Assay of Vibrio cholerae by Polymerase Spiral Reaction (PSR) in Emergency Situations

Abstract

Background: The purpose of this survey was to develop a novel and rapid isothermal nucleic acid based detection assay of Vibrio cholerae by polymerase spiral reaction (PSR) in emergency situations.

Methods: The current study was conducted in Baqiyatallah University of Medical Sciences, Tehran, Iran in 2021. The conserved ctxA gene sequence of V. cholerae was used as a target of designed two pairs of primers. Amplification of nucleic acids performed under isothermal temperature of 65 oC in 55 min by using Bst DNA polymerase. PSR amplified products were real-time visualized under UV transilluminator and also on agarose gel electrophoresis.

Results: Seven non- V. cholerae bacteria were negative for detection, which indicated the specificity of PSR assay was 100%. A 10- fold serial dilution of V. cholerae genomic DNA was subjected to conventional polymerase chain reaction (PCR) and real-time PCR to compare their sensitivities with PSR. The detection limit of PSR was 3 × 10-5 ng/ μL within 60 min, which 100-fold higher than that of PCR (3 × 10-3 ng/μL), but the sensitivity of real-time PCR was found as same as it.

Conclusion: The PSR assay developed in this study can provide a simple, cost-effective, rapid, and precise diagnosis technique in endemic cholera outbreaks, especially in low-income with limited access provinces.

 

1. Chowdhury FR, Nur Z, Hassan N, von Seidlein L, Dunachie S (2017). Pandemics, pathogenicity and changing molecular epidemiology of cholera in the era of global warming. Ann Clin Microbiol Antimicrob, 16:10.
2. Louhrasby V, Ghayaz F, Mirhosseini SA, Golmohammadi R (2020). A systematic review of 40 years of distribution and antimicrobial resistance of Vibrio cholerae in Iran with future perspective. Rev Med Microbiol, 31:183-190.
3. Das B, Pazhani GP, Sarkar A, Mukhopadhyay AK, Nair GB, Ramamurthy T (2016). Molecular evolution and functional divergence of Vibrio cholerae. Curr Opin Infect Dis, 29:520-527.
4. Murray PR, Rosenthal KS, Pfaller MA (2020). Medical Microbiology E-Book. ed. Elsevier Health Sciences.
5. Keddy KH, Sooka A, Parsons MB, Njanpop-Lafourcade B-M, Fitchet K, Smith AM (2013). Diagnosis of Vibrio cholerae O1 infection in Africa. J Infect Dis, 208:S23-S31.
6. Alam M, Hasan NA, Sultana M, et al (2010). Diagnostic limitations to accurate diagnosis of cholera. J Clin Microbiol, 48:3918-3922.
7. Chu LC, Goggins MG, Fishman EK (2017). Diagnosis and detection of pancreatic cancer. Cancer J, 23:333-342.
8. Liu W, Dong D, Yang Z, Zou D, Chen Z, Yuan J, Huang L (2015). Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method. Sci Rep, 5:12723.
9. Liu W, Zou D, He X, et al (2018). Development and application of a rapid Mycobacterium tuberculosis detection technique using polymerase spiral reaction. Sci Rep, 8:3003.
10. Okada K, Chantaroj S, Taniguchi T, et al (2010). A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples. Diagn Microbiol Infect Dis, 66:135-139.
11. Bennett JE, Dolin R, Blaser MJ (2020). Bennett’s principles and practice of infectious diseases. Ninth Edition, Canada Elsevier
12. Zhang X, Lu Y, Qian H, et al (2020). Non-O1, Non-O139 Vibrio cholerae (NOVC) Bacteremia: Case Report and Literature Review, 2015–2019. Infect Drug Resist, 13:1009.
13. Mondiale de la Santé O, Organization WH (2017). Cholera vaccines: WHO position paper–August 2017–Vaccins anticholériques: Note de synthèse de l’OMS–août 2017. Wkly Epidemiol Rec, 92:477-498.
14. Jameel SK, Shafek MA, Abdulmohsen AM, Mohamed NS, Naji SR, Mohammed TT (2016). The Isolation of Vibrio cholera and Other Enteric Bacteria with Molecular Characterization of Vibrio cholera during the Outbreak of Baghdad/Iraq in 2015. Adv Microbiol, 6:699.
15. Jiang X, Dong D, Bian L, et al (2016). Rapid Detection of Candida albicans by polymerase spiral reaction assay in clinical blood samples. Front Microbiol, 7:916.
16. Das A, Kumar B, Chakravarti S, et al (2018). Rapid visual isothermal nucleic acid-based detection assay of Brucella species by polymerase spiral reaction. J Appl Microbiol, 125:646-654.
17. Gupta V, Chakravarti S, Chander V, et al (2017). Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA. Arch Virol, 162:1995-2001.
18. Dong D, Zou D, Liu H, et al (2015). Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China. Front Microbiol, 6:1100.
19. Soleimani M, Shams S, Majidzadeh-A K (2013). Developing a real-time quantitative loop-mediated isothermal amplification assay as a rapid and accurate method for detection of Brucellosis. J Appl Microbiol, 115:828-834.
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IssueVol 51 No 6 (2022) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijph.v51i6.9682
Keywords
Vibrio cholerae Polymerase spiral reaction (PSR) Isothermal nucleic acid amplification Isothermal
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How to Cite
1.
Louhrasby V, Mirhosseini SA, Golmohammadi R. A Novel and Rapid Isothermal Nucleic Acid Based Detection Assay of Vibrio cholerae by Polymerase Spiral Reaction (PSR) in Emergency Situations. Iran J Public Health. 2022;51(6):1364-1370.