Iranian journal of public health · 2022
A Novel and Rapid Isothermal Nucleic Acid Based Detection Assay of Vibrio cholerae by Polymerase Spiral Reaction (PSR) in Emergency Situations.
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Louhrasby Vahid, Mirhosseini Seyed Ali, Golmohammadi Reza
Аннотация
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 °C 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.
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 °C 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.