基于HSP90基因的羊吕氏泰勒虫PCR检测方法的建立与评价OA

Theileria luwenshuni(T.luwenshuni)is a blood protozoan that severely impacts the sheep industry,and es-tablishing a rapid and accurate detection method is crucial for its prevention and control.To develop a PCR assay target-ing the heat shock protein 90 gene(HSP90)of T.luwenshuni as a novel marker,this study designed specific primers based on the HSP90 gene sequence.Reaction conditions were optimized via gradient PCR,and the method's sensitivity,specificity,and repeatability were systematically evaluated.The results showed that the optimal annealing temperature and time for the PCR detection method established in this study were 60.5℃and 30 s,respectively.Performance evalua-tion demonstrated that the method had high sensitivity,with a minimum detection limit of 32.4 pg/µL of genomic DNA(over 3 times more sensitive than the 18S rRNA target).The method exhibited strong specificity,showing no cross-reaction with DNA from Cysticercus pisiformis,Echinococcus multilocularis,Toxoplasma gondii,or Eimeria spp.It also displayed good repeatability,yielding consistent results across inter-and intra-assay tests.Detection of field-collected sheep blood samples revealed a positivity rate of 27.78％(18 samples),and sequencing confirmed the amplified prod-ucts as T.luwenshuni.This study successfully established a PCR method for detecting T.luwenshuni based on the HSP90 gene.This efficient,sensitive,specific,and reliable assay not only provides a new effective tool for epidemio-logical investigation and precise diagnosis but also lays a solid foundation for future research into HSP90 gene function and the development of targeted drugs,given its critical role in Theileria luwenshuni stress survival and pathogenicity.