The Study Of Rapid Detection Of Clostridium Perfringens By Real-Time Quantitative PCR

ObjectiveGas gangrene is a severe bacterial infection that produces gas in tissues in gangrene. It is a deadly form of gangrene usually caused by Clostridium perfringens. It is a medical emergency. Gangrenous infection by soil-borne bacteria was common in the combat injuries of soldiers, due to non-sterile field surgery and the basic nature of care for severe projectile wounds.If Gas gangrene occur in war or emergencies, it requires a technology which is the large amount of sample testing, hazard assessment and quantitative accuracy, but the current technology can not solve the problems. The purpose of the study is established a real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) method for specific, sensitive and rapid detection of Clostridium perfringens.The method is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a Clostridium perfringens DNA molecule.It can reduce disability rate and death of trauma patients in war and emergency situations, and improve patient quality of life in peacetime. Explore antimicrobial susceptibility of Clostridium perfringens, in the hope that choice of antibiotics as soon as possible to minimize the incidence of amputation and antibiotics abuse.MethodsThe gene sequences of Clostridium perfringens downloaded from the genebank was used as a template, a pair of primers and probe were designed depending on the conserved region of the16s rDNA gene from Clostridium perfringens. The primers, probe and the reactive condition were optimized to improve the reaction conditions and reaction parameters. To create standard curves with known amounts of standard plasmid. The standard plasmid is the PCR amplified products ligated to pMD19-T.The Culture-negative specimens were prepared to join the standard strains of Clostridium perfringens bacteria into a simulated specimen. Collection of clinical specimens and compared with anaerobic culture, and detection simulated specimen verify the specificity, sensitivity and feasibility of clinical application of this system. Select10kinds of antibiotics according to CLSI, and approach the drug sensitivity test for Clostridium perfringens.ResultsA simple and rapid real-time PCR assay was established for quantification of Clostridium perfringens in wounds secretion samples. This detection system can be completed within three hours.The established FQ-PCR methed showed a high specificity for Clostridium perfringens, and no cross reaction with53kinds of aerobic and6kinds of anaerobic bacteriaThe sensitivity of the assay was evaluated with purified Clostridium perfringens (ATCC13124DNA)genomic DNA. With pure DNA, the reproducible limit of detection was approximately9×102cfu/ml of Clostridium perfringens genomic DNA.The coefficient of variation(CV)for the four spiked series were1.57,1.21,0.6,0.81,accord with standard preferred for moderately laboratory by WHO. For558clinical specimens, there was no significant difference between PCR and bacterial culture and Kappa value is0.983. There are two Clostridium perfringens strains resistance of Clindamycin. Results are reported as susceptible for other drugs which CLSI recommend.ConclusionThe FQ-PCR methed established is correspond with the traditional anaerobic culture but detection time greatly reduce than traditional methods. No cross reaction with other aerobic and anaerobic bacterium of wound secretions, it is specificity.The real-time quantitative PCR assay is a sensitive and specific technique for usual monitoring and emergent examination of Clostridium perfringens. In this study, the technique can do gene amplification, hybridization and quantitative, able to make rapid and accurate identification and susceptibility report on wartime, as far as possible to reduce amputation and death occurs. All drugs are sensitivity for Clostridium perfringens but clindamycin. So,the clindamycin is not the first choise for Clostridium perfringens.