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Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Clostridium perfringens in meat.

ABSTRACT: C. perfringens is a widespread foodborne pathogen and one of the major concerns in the meat industry. There is a need for a simple, rapid and equipment free detection system for C. perfringens as conventional anaerobic culture method is labour and resource intensive. Here, we applied a novel polymerase spiral reaction phenomenon to develop and evaluate an assay for effortless and visual detection of C. perfringens in meat foods employing pork as a representative model. Specificity of the assay was determined using 51 C perfringens and 20 non- C. perfringens strains. Analytical sensitivity of the developed test was 80 fg DNA per tube indicating 100 times more sensitivity than end-point PCR assay. The detection limits were 980 CFU/g and 9.8 × 10⁴ CFU/g of pork for PSR and PCR assays, respectively. The operation time of the PSR assay including DNA extraction was 120 min. The developed PSR assay was accurate and effective in comparison to culture method, in detecting C. perfringens in 38 of 74 pork samples. Therefore the specificity, sensitivity, negative predictive value, positive predictive value and accuracy rate of the developed PSR assay were 100%. The developed PSR assay is easy to perform, rapid, affordable, permitting sophisticated-equipment free amplification and naked eye interpretation. This is the initial report in which the PSR assay was optimized for the detection of C. perfringens.

SUBMITTER: Milton AAP

PROVIDER: S-EPMC7810786 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:West Nile virus (WNV) is a mosquito-borne virus of public health importance. Currently, there is no FDA approved vaccine available against WNV infection in humans. Therefore, the early diagnosis of the WNV infection is important for epidemiologic control and timely clinical management in areas where multiple Flaviviruses are endemic. The present study aimed to develop reverse transcription polymerase spiral reaction (RT-PSR) assay that rapidly and accurately detects the envelope (env) gene of WNV. RT-PSR assay was optimized at 63°C for 60 min using real-time turbidimeter or visual detection by the addition of SYBR Green I dye. The standard curve for RT-PSR assay was generated using the 10-fold serial dilutions of in vitro transcribed WNV RNA. To determine the detection limit of RT-PSR assay, an amplified product of conventional RT-PCR was in vitro transcribed as per standard protocol. The detection limit of the newly developed RT-PSR assay was compared with that of conventional RT-PCR and CDC reported TaqMan real-time RT-PCR using a serial 10-fold dilution of IVT WNV RNA. The detection limit of RT-PSR was found to be 1 RNA copy, which is 100-fold higher than that of conventional RT-PCR (100 copies). This suggests that RT-PSR assay is a valuable diagnostic tool for rapid and real-time detection of WNV in acute-phase serum samples. The assay was validated with a panel of 107 WNV suspected human clinical samples with signs of acute posterior uveitis and onset of febrile illness. Out of 107 samples, 30 were found positive by RT-PSR assay. The specificities of the selected primer sets were established by the absence of cross-reactivity with other closely related members viruses of the Flaviviruses, Alphaviruses, and Morbilliviruses groups. No cross-reactivity was observed with other viruses. To best of our knowledge, this is the first report describing the RT-PSR assay for the detection of RNA virus (WNV) in clinical samples. RT-PSR is a high throughput method and more than 30 reactions can be run at once in real-time turbidimeter. PSR assay has potential to be used for a rapid screening of large number of clinical samples in endemic areas during an outbreak.

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Development of a novel polymerase spiral reaction (PSR) assay for rapid and visual detection of Clostridium perfringens in meat.

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