ESTABLISHMENT AND APPLICATION OF A MULTIPLEX PCR DETECTION METHOD FOR THREE COMMON PATHOGENS CAUSING DIARRHEA IN YAKS
10.3969/j.issn.1005-0507.2025.03.003
- VernacularTitle:牦牛腹泻3种常见病原体多重PCR检测方法的建立与应用
- Author:
Yao PAN
1
;
Jing ZHANG
;
Mei-Jun JIN
;
Ling-Xiang XIN
;
Hai-Yue XIAO
;
Yan LIU
;
Wen-Sheng YAO
;
Chuan CHENG
;
Li-Li HAO
;
Lan LAN
;
Liang-Quan ZHU
Author Information
1. 四川省甘孜藏族自治州畜牧业科学研究所,四川康定 626000
- Keywords:
Yak calves;
Cryptosporidium;
Eimeria;
Parvovirus;
Multiplex PCR
- From:
Acta Parasitologica et Medica Entomologica Sinica
2025;32(3):146-152
- CountryChina
- Language:Chinese
-
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.
