微小隐孢子虫PCR检测方法的建立与应用OA

This study aimed to establish a PCR method for the detection of Cryptosporidium parvum.Based on the GP60 gene sequence of Cryptosporidium parvum downloaded from GenBank,a pair of specific primers was designed targeting a highly conserved region.The PCR method was established through program optimization,and its sensitivity,specificity,repeatability,and clinical sample detection performance were evaluated.The results showed that the established PCR method could amplify a specific 921 bp fragment of Cryptosporidium parvum,consistent with the expected size.No cross-reactions were observed with enterotoxigenic Escherichia coli K99,Clostridium perfringens,Eimeria sp.,bovine rotavirus,and bovine coronavirus.After serial dilution of positive samples,the minimum detection concentration was 0.243 mg/L.Intra-assay and inter-assay results demonstrated good repeatability and stability.Using this method,411 fecal and anal swab samples from diarrheic cattle collected in Heilongjiang Province were tested,and 141 samples were positive for Cryptosporidium parvum,with an overall positive rate of 34.31%.The study indicates that the established method has good specificity,sensitivity,and repeatability,and is well suited for the detection of clinical samples.