| Clostridium perfringens(C.perfringens)is a Gram-positive anaerobic bacterium that is widely present in the environment,including contaminated water,food and soil.When the body’s gastrointestinal flora is disturbed and the temperature drops sharply,it can multiply and produce different toxins,causing human and animal gas gangrene,food poisoning,enteritis,enterotoxemia and other diseases,seriously endangering the health of humans and animals.It not only causes serious economic losses to farmers,affects the development of animal husbandry,but also poses a threat to public safety.The typing of C.perfringens is complex,and specific diagnosis is an important link in the prevention and treatment of the disease.Therefore,in this study,five toxins α,β,ε,ι and cpe of C.perfringens were used as target genes.Specific primers and TaqMan probes were designed to establish a multiple TaqMan fluorescence quantitative PCR(qPCR)detection method that can detect five toxin genes,and an epidemiological investigation of C.perfringens in cattle and sheep in some areas of Ningxia was carried out.The results of the study are as follows.1.Establishment of multiplex TaqMan qPCR assay for C.perfringens.Based on the sequences of C.perfringens α,β,ε,ι and cpe toxin genes in the NCBI database,we designed specific primers and TaqMan probes,and established of multiplex TaqMan qPCR assay for the simultaneous detection of five toxin genes by constructing standard positive plasmids,optimizing reaction conditions,constructing standard curves,and performing specificity,sensitivity,reproducibility and clinical sample detection tests.The results showed that the established multiplex TaqMan qPCR assay had good linearity of its amplification curve with R2>0.99 and 90%<E<110%;with high specificity and no cross-reactivity with different pathogens;the method had high sensitivity and the lower limit of detection for all five positive plasmids was 102 copies/μL,which was 10～100 times more sensitive than that of the common PCR;the method had good reproducibility,and the coefficient of variation was less than 2%;using the method established in this experiment and the established ordinary PCR for 50 fecal samples and 40 bacterial fluid samples,the qPCR detected 7 more a toxins and 5 more ε toxins on the basis of the ordinary PCR.2.Investigation of C.perfringens toxin genes in cattle and sheep in Ningxia regionA total of 623 fecal samples of diarrhea from cattle and sheep farms in five cities in Ningxia region were collected,tested by molecular biology,and analyzed for toxin typing based on Ct values.The results showed that 354 positive samples of C.perfringens were identified,the positive rate was as high as 56.82%,and 511 strains of C.perfringens were speculated.The positive rate of C.perfringens in sheep feces was 58.94%(201/341),and the positive rate of C.perfringens in cow feces was 54.26%(153/282).The toxin genes were identified as a toxin 56.82%(354/623),βtoxin 9.03%(32/354),ε toxin 43.98%(157/354),ι toxin 33.90%(12/354),and cpe toxin 33.62%(119/354).According to the Ct value,281 strains of C.perfiingens type A(54.99%),12 strains of type B(2.35%),19 strains of type C(3.72%),146 strains of type D(28.57%),7 strains of type E(1.37%)and 46 strains of type F(9.00%)were inferred.There are some differences in the proportion of toxin genes in farms in different regions;the incidence of C.perfringens in cattle and sheep at different months of age was mainly concentrated in 0～6 months of age,with an incidence rate of 39.55%.The incidence of C.perfringens in cattle and sheep between different seasons was mainly concentrated in summer,with an incidence of 34.46%.The results showed that the multiplex TaqMan qPCR detection method established in this experiment could be used for the detection of toxin genes of C.perfringens.The investigation of the prevalence of toxin genes showed that there were different types of C.perfringens infection in Ningxia.The results provided a scientific basis for the diagnosis and prevention of C.perfringens in Ningxia. |
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