寄生虫与医学昆虫学报2025，Vol.32Issue(3)：146-152,7.DOI:10.3969/j.issn.1005-0507.2025.03.003
牦牛腹泻3种常见病原体多重PCR检测方法的建立与应用
ESTABLISHMENT AND APPLICATION OF A MULTIPLEX PCR DETECTION METHOD FOR THREE COMMON PATHOGENS CAUSING DIARRHEA IN YAKS
摘要
Abstract
Objective To establish a rapid,sensitive,and specific multiplex PCR detection method for the simultaneous detection of Cryptosporidium,Eimeria,and bovine parvovirus.Methods Specific primers targeting the SSU rRNA genes of Cryptosporidium and Eimeria,as well as the VP2 gene of bovine parvovirus were designed and the corresponding recombinant plasmid standards were constructed.To establish the multiplex PCR method,the reaction conditions were optimized using temperature gradient PCR and single-variable control methods.The sensitivity,specificity,reproducibility,and clinical application of the protocol were evaluated.Results The optimal annealing temperature was found to be 60.5℃,and the forward and reverse primer concentrations were determined to be 0.2 μmol/L for Eimeria,and 0.4 μmol/L for Cryptosporidium and bovine parvovirus.The assay demonstrated high sensitivity,with detection limits of 243,260,and 3 110 copies for the recombinant plasmid standards of Cryptosporidium,Eimeria,and bovine parvovirus,respectively.Specificity testing showed no cross-reactivity with ten common bovine pathogens,including Salmonella,bovine viral diarrhea virus,and bovine rotavirus.Consistent intra-and inter-batch results confirmed the strong reproducibility of the method.Clinical application to 81 diarrhea samples from various regions in the Ganzi Prefecture,Sichuan,revealed positivity rates of 18.52%(15/81)for Cryptosporidium,34.57%(28/81)for Eimeria,and 18.52%(15/81)forbovineparvovirus,withamixedinfectionrateof3.7%(3/81).Conclusions Themultiplex PCR method established in this study offers a reliable tool for differential diagnosis and epidemiological investigation of the three common diarrheal pathogens in yaks.关键词
牦牛/隐孢子虫/球虫/细小病毒/多重PCRKey words
Yak calves/Cryptosporidium/Eimeria/Parvovirus/Multiplex PCR分类
基础医学引用本文复制引用
潘瑶,蓝岚,朱良全,张晶,金美君,辛凌翔,肖海月,刘燕,姚文生,程川,郝力力..牦牛腹泻3种常见病原体多重PCR检测方法的建立与应用[J].寄生虫与医学昆虫学报,2025,32(3):146-152,7.基金项目
国家重点研发计划项目(2022YFD1800703) （2022YFD1800703）
四川省科技厅苗子工程重点项目(2023JDRC0121) （2023JDRC0121）
