Development And Evaluation Of Real-time PCR Methods For Toxigenic Strains And Point Mutation Of Moxifloxacin Resistant GyrA Gene In Clostridioides Difficile

Objectives:To develop a novel multiplex real-time PCR method with high sensitivity,specificity and repeatability,detecting toxigenic Clostridioides difficile directly from stool samples and determining the toxigenic type of C.difficile accurately.In addition,to develop a duplex Real-time PCR method for detecting the point mutation of the moxifloxacinresistant gyrA gene in C.difficile and determining whether the point mutation occurred in the resistant gene,and then predicting whether the samples are resistant to moxifloxacin.Methods:Three toxin genes tcdA,tcdB,cdtB of C.difficile were used as target genes by multiplex real-time PCR method,primers and Taq-Man probes were designed based on their conserved sequences,the PCR products were purified and plasmid standards were constructed,standard curves were made after gradient dilution.The detection limit of this method for pure bacterial DNA were determined and compared with PCR.The specificity of this method was verified by other common intestinal pathogens(Escheric/hia coli,Enterococcus Faecom,Enterococcus faecalis,Clostridium botulinum,Clostridium perfringen,Bacterooides fragilis)and standard strains of toxigenic C.difficile.The repeatability of this method was verified by repeated tests between different batches.The diluted bacteria were added into the fecal samples of healthy people to construct the simulated fecal samples,the detection limit of DNA in fecal samples was determined.The pure bacterial DNA of 69 C.difficile isolates and the DNA of 74 fecal samples were detected by this method and the results of the gold standard(toxin-producing culture)were used to evaluate the performance of this method.The specific primers of gyrA,different TaqMan-MGB probes targeting moxifloxacin sensitive strains with ACT and resistant strains with ATT mutation site were designed respectively by duplex Real-time PCR method.This PCR system was further evaluated and improved.The sensitivity,specificity and repeatability of the method were verified.The gyrA genes of 73 clinical isolates of C.difficile were amplified by PCR and the type of mutation sites was identified by sequencing.At the same time,the established duplex Realtime PCR method was used to detect the 73 clinical isolates,and the consistency of the two methods was evaluated.Results:The annealing temperature and GC content of primers designed for C.difficile meet the requirements,after optimizing the system and parameters,the amplification efficiency of the three target genes could reach 102%,103%and 102%,respectively.The lower limit of tcdA gene detection by PCR was 102 copies/μL,and the sensitivity of Real-time PCR(101 copies/μL)was 10 times that of PCR.The lower limit of tcdB gene detection by PCR was 103 copies/μL,and the sensitivity of Real-time PCR(100 copies/μL)was 1000 times that of PCR.The lower limit of cdtB gene detection by PCR was 102 copies/μL,and the sensitivity of Real-time PCR(100 copies/μL)was 100 times that of PCR.There were no amplification curves in the detection of other common intestinal pathogens,and the detection of C.difficile standard strains were consistent with the results of toxin spectrum.The intra-batch and inter-batch variation coefficients were less than 3%.The lower limits of DNA detection for stool samples were 100,103 and 102 CFU/g,respectively.The sensitivity and specificity of 69 pure bacteria DNA were 100%compared with the results of gold standard.The sensitivity and specificity of DNA from 74 fecal samples were 96.49%(55/57)and 94.12%(16/17),respectively.The lower limit of detection for moxifloxacin resistant strains and sensitive strains were 4.11 copies/μL and 5.14 copies/μL respectively for the duplex Real-time PCR for moxifloxacin resistant gyrA gene point mutation.The amplification of 6 common intestinal pathogens was negative,and the detection specificity of the method was 100%.The intrabatch and inter-batch variation coefficients of the three gradients were all less than 5%.The consistency of Real-time PCR results with the sequencing can reach k=0.97(p<0.05).Conclusion:In this study,a Taq-Man multiplex Real-time PCR method for detecting C.difficile directly from fecal samples was established.It provides a rapid detection method for tcdA,tcdB,cdtB genes without depending on special instruments and it can accurately identify C.difficile.This method demonstrated high sensitivity,specificity and repeatability of detecting toxigenic C.difficile from pure strain and fecal samples.the method can be equipped with POCT(Point-of-Care Testing)for rapid detection and identification of clinical CDI cases.Due to the small sample capacity,the performance of this method needs to be verified by more clinical specimens.In addition,the duplex Realtime PCR method was established for moxifloxacin-resistant gyrA gene point mutation of C.difficile with high sensitivity,specificity and repeatability.And it is efficient and accurate for the identification of this resistant gene.