Rapid Diagnostic Strategies Research Of Norovirus Infection And Nosocomial Clostridium Difficle Infection(CDI)

Norovirus is a common cause of outbreaks of acute gastroenteritis.Norovirus infection is associated with mortality among children in developing countries,as well as vulnerable groups with low immunity and in older patients.The wide range of mutations observed between different genotypes of Norovirus may affect diagnosis,as the sites identified by antibodies used in serological testing may change due to antigen drift,and untranslated mutations in the genome may also affect the ability of molecular diagnostics to detect mutations in primer binding sites.Norovirus infection can cause gastrointestinal symptoms,mainly diarrhea,and vomiting.Clostridium difficle infection(CDI)is the main cause of gastrointestinal disease outbreaks in hospitals.The pathogenic mechanism of C.difficile is attributed to specific toxins(toxin A,toxin B,and binary toxins)that cause pseudomembranous colitis and antibiotic-related diarrhea.Although commercial kits are available for the extraction of pathogenic bacteria from stool samples,the pretreatment of clinical stool samples and the C.difficile DNA extraction process has not yet been standardized.Taq Man real-time PCR is widely used for the detection of pathogenic microorganisms in clinical samples as it boasts high sensitivity and specificity in addition to short operation time.In the present paper,a fast and convenient method for the extraction of C.difficile DNA from clinical stool samples is proposed.Based on Taq Man real-time PCR,a duplex detection primer and a Taq Man probe for C.difficile were designed and confirmed to be effective.The integrated cartridge was used to extract the DNA of two pathogens.The specific research contents of this paper are as follows:1.Pretreatment of stool samples and optimization of DNA extraction stepsStool samples were pretreated with a phosphate-buffered saline buffer using various techniques,including dissolving,shaking,and centrifuging,to remove impurities and retain bacteria.The reagentvolume,shaking time,centrifugation strength,and centrifugation time was varied until the most suitable pretreatment protocol for stool samples was determined.Six commercially available DNA extraction kits were compared and comprehensively evaluated based on various parameters,including extraction cost,operation time,number of operations required,and extraction effect(downstream PCR application),to optimize DNA extraction while ensuring efficacy.Of the extraction kits evaluated,the QG-S kit had the highest cost and the shortest operating time;the SG-B kit had the lowest cost and the longest operating time.The concentration of DNA extracted using the AS-B kit was the highest(approximately83.14 ± 24.95 ng/μL);the concentration of DNA extracted using the SG-B kit was the lowest(approximately 8.24 ± 0.08 ng/μL).With the SG-B kit and the TL-B kit,the optical density value(0.86 ± 0.01 and 1.37± 0.01,respectively)of the DNA extracted deviated from the normal range.DNA extracted using the TG-B kit was contaminated with proteins.Real-time PCR analysis showed that the QG-S kit provided the highest experimental stability and reproducibility.The magnetic beads from the LS-B kit and the lysis buffer from the AS-B kit can be used to quickly extract C.difficile DNA from stool samples,thereby optimizing the extraction step.2.Design of C.difficile duplex detection primers and Taq Man probes and simulation automated detection with cartridgeTwo methods were used to design C.difficile duplex real-time PCR detection primers and Taq Man probes.The first method involved designing duplex detection primers and then manually designing and confirming Taq Man probes based on the amplicon regions of the primers.The second method entailed first analyzing target sequence information,then intercepting fragments with higher CG content for primer and probe design,and finally synthesizing the primers for a specificity test.After evaluating the best specific primer combination,corresponding probes were synthesized and confirmed.The application of the probes designed in the first method failed,but one set of primer and probe combinations in the second method could be applied in duplex real-time PCR.This resulted in improved sensitivity but slightly reduced fluorescence signalintensity.Norovirus standard and C.difficile stool samples were simulated and extracted automatically in an integrated test cartridge.The standard curve of the Norovirus standard had a slope of approximately-3.4 and an amplification efficiency of approximately 2,indicating that the overall amplification efficiency was correspondingly high;meanwhile,the standard curve of the C.difficile sample had a slope of approximately-3.0 and an amplification efficiency of 2.13,indicating that the amplification efficiency was not ideal.Therefore,the extraction efficiency for the Norovirus standard was higher than that for the C.difficile sample.