Development of a dry reagent-based triplex PCR for the detection of toxigenic and non-toxigenic XXXX .

@entity3081 @entity7970 @entity1838
@entity1838 has caused severe outbreaks of @entity3081 worldwide with thousands of recorded deaths annually. Molecular diagnosis for @entity3081 has become increasingly important for rapid detection of @entity3081 as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for @entity3081 which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of @entity1838 based on the @entity3081 toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of @entity7970 , which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100% sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 7 months at 24   C. The limit of detection of the thermostabilized triplex PCR assay was 2 10(4) c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic @entity1838 which requires minimal pipetting steps and is cold chain-free.
