Establishment And Evaluation Of A Nucleic Acid Amplification Based Detection Method For Clostridium Difficile

Clostridium difficile is an obligate anaerobic Gram-positive bacillus,which generally parasitize in the human gut.Clostridium difficile infection usually occurs after the long-term or adverse use of antibiotics causes intestinal flora imbalance,and manifests as diarrhea,abdominal pain,leukocytosis and other symptoms.The detection methods of Clostridium difficile mainly include fecal culture,cytotoxicity assay and glutamate dehydrogenase assay.However,these methods are time-consuming,complex and low sensitivity.Nucleic acid amplification technology has attracted wide attention due to its rapid reaction and high sensitivity.Since the development of technology,nucleic acid amplification technology has been continuously developed and improved.At present,the common nucleic acid amplification techniques include real-time quantitative polymerase chain reaction and loop-mediated isothermal amplification.The former belongs to variable temperature amplification and requires precision instruments,and the latter has complex primer design and is prone to false positive or false negative results.Therefore,the development of a novel,rapid,simple,accurate and sensitive nucleic acid amplification based detection method for Clostridium difficile is of great significance for the timely diagnosis of Clostridium difficile infection,prevention and control of intestinal infectious diseases caused by Clostridium difficile.Objective:The polymerase spiral reaction technique exploits the strand-displacement activity of Bst DNA polymerase to achieve nucleic acid amplification with a pair of primers and one enzyme in an isothermal environment.In view of the lack of rapid and sensitive on-site detection tools for Clostridium difficile,the aim of this study is to establish a polymerase spiral isothermal amplification method for rapid visual qualitative detection of Clostridium difficile in grass-root units or field emergencies.Droplet digital PCR reaction technology is based on PCR reaction,which realizes highly sensitive detection and quantification of nucleic acid through the microdrop treatment of samples.The aim of this study is to develop a droplet digital PCR method for accurate and sensitive quantitative detection of Clostridium difficile in complex samples in the clinical laboratory environment.Methods:1.Rapid visual detection of Clostridium difficile based on polymerase spiral reaction technology.Firstly,Clostridium difficile was purified and cultured,and the bacterial nucleic acid was extracted by boiling method.The main primers and acceleration primers of the polymerase spiral reaction were designed according to the specific toxin tcdB gene of Clostridium difficile.The polymerase spiral reaction system was established and optimized in terms of primers and reaction temperature.Hydroxynaphthol blue indicator dye was introduced into the system,and the indicator dye combined with magnesium ions turned purple,while the loss of magnesium ions made the system color blue.The color changes of positive and negative samples were observed by naked eyes,and a rapid visual detection method for Clostridium difficile was established.The sensitivity,specificity,detection limit and clinical applicability of the method were evaluated by simulating feces samples and clinical isolates.2.Quantitative detection of Clostridium difficile based on droplet digital PCR.In this method,the primers and probes for microdroplet digital PCR and real-time fluorescence quantitative PCR were evaluated and synthesized based on the tcdB gene,a specific toxin of Clostridium difficile.Firstly,the microdroplet digital PCR reaction system was established and optimized using plasmid DNA and pure bacterial DNA,and the fluorescent signal of amplified products was detected.The quantitative detection was achieved by one-dimensional distribution of negative and positive droplets.The feasibility of the established method was evaluated in simulated feces samples and clinical isolates.Results:1.Rapid visual detection of Clostridium difficile based on polymerase spiral reaction technology.(1)A pair of main primers and a pair of acceleration primers were designed to accelerate the amplification of tcdB gene,Then the primers and reaction temperature that had the greatest effect on the amplification efficiency of the reaction were selected and optimized,and 64℃ was selected as the best reaction temperature and primer 1 was selected as the best primer,containing a pair of master primers FIP and BIP,and a pair of acceleration primers LF and LB,at which time the amplification efficiency was the highest.(2)The sensitivity of this method for the detection of Clostridium difficile genomic DNA was up to 150 fg/μL,and the detection limit of simulated feces samples was as low as 2×101 CFU/mL.The whole process could be completed within 60 minutes,and the specificity was good.To provide technical support for rapid on-site screening of Clostridium difficile infection samples.The results of detection of 5 clinical Clostridium difficile isolates were consistent with gene sequencing,conventional PCR,isolation and culture identification method,which could also be applied to the detection of simulated stool samples and clinical isolates.2.Quantitative detection of Clostridium difficile based on droplet digital PCR.(1)The primers and probes of microdroplet digital PCR reaction were evaluated and synthesized according to the tcdB gene of Clostridium difficile.The droplet digital PCR reaction system was established and optimized.Under the annealing temperature of 56℃,the obvious layer between positive and negative droplets could be achieved,and the amplification effect was the best.For the detection of Clostridium difficile in plasmid samples,the sensitivity of droplet digital PCR was as low as 8.856 copies/reaction and the linear relationship was stable(R~2>0.99).Compared with realtime fluorescence quantitative PCR,the performance of droplet digital PCR was more stable at low concentration of pathogenic bacteria.(2)For the detection of Clostridium difficile in simulated feces samples,the linear range of droplet digital PCR was 1×106～1×10~1 CFU/mL,with high sensitivity,strong stability and wide detection range.In the detection of clinical isolates,the positive results of droplet digital PCR were consistent with those of gene sequencing and electrophoresis,which proved that droplet digital PCR could be used for quantitative detection of Clostridium difficile in clinical laboratories.Conclusion:(1)A polymerase spiral reaction method was established to achieve visual and qualitative detection of Clostridium difficile in feces by hydroxynaphthol blue dye.(2)A droplet digital PCR method was established to achieve quantitative detection of Clostridium difficile in feces.(3)The two detection methods for Clostridium difficile have good specificity,high sensitivity and wide detection range,which are suitable for point-of-care detection and clinical laboratory detection,respectively.