Article(id=1297139318028530626, tenantId=1146029695717560320, journalId=1296801528146919473, issueId=1297139249468433079, articleNumber=null, orderNo=null, doi=10.12377/1671-4393.26.05.07, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=null, receivedDateStr=null, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1787191713100, onlineDateStr=2026-08-20, pubDate=1779638400000, pubDateStr=2026-05-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1787191713100, onlineIssueDateStr=2026-08-20, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1787191713100, creator=13701087609, updateTime=1787191713100, updator=13701087609, issue=Issue{id=1297139249468433079, tenantId=1146029695717560320, journalId=1296801528146919473, year='2026', volume='', issue='5', pageStart='2', pageEnd='144', issueExtLink='null', onlineDate='null', pubDate='1779638400000', pubDateStr='2026-05-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1787191696754, creator='13701087609', updateTime=1787192231935, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1297141494226383122, tenantId=1146029695717560320, journalId=1296801528146919473, issueId=1297139249468433079, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1297141494226383123, tenantId=1146029695717560320, journalId=1296801528146919473, issueId=1297139249468433079, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=41, endPage=46, ext={EN=ArticleExt(id=1297139319630754755, articleId=1297139318028530626, tenantId=1146029695717560320, journalId=1296801528146919473, language=EN, title=Establishment and Application of a Multiplex qRT-PCR Method for Detecting Calf Diarrhea Disease, columnId=1297134042869158672, journalTitle=China Dairy, columnName=DISEASE PREVENTION, runingTitle=null, highlight=null, articleAbstract=
[Objective] To establish a multiplex qRT-PCR method for detecting calf diarrhea disease, aiming to simultaneously and rapidly detect multiple pathogens that cause diarrhea in calves.
[Method] This study searched for conserved gene sequences of various pathogens causing diarrhea in calves as target genes, designing specific primers and probes, constructing plasmid standards, optimizing primer concentrations and annealing temperatures, and testing their sensitivity, specificity, and clinical sample validation.
[Result] A multiplex qRT-PCR method for detecting calf diarrhea disease was successfully established using 103 copies/μL plasmid as the standard.The reaction system consisted are 10 μL 2 × Fast-Start Universal SYBR Green Master, 0.5 μL upstream primer, 0.5 μL downstream primer, 1 μL template, 8 μL sterilized ultrapure water, and a total reaction volume of 20 μL. The reaction conditions are: 95 ℃ for 3 minutes; 94 ℃ for 20 seconds, 56 ℃ for 15 seconds, 72 ℃ for 30 seconds, 36 cycles; 72 ℃ for 5 minutes. The detection sensitivity is 1 copy/μL, and there is no cross-reactivity with other pathogens. The intra-group and inter-group coefficients of variation are both less than 2%. Among 315 clinical fecal samples of diarrheal calves, a total of 248 are detected as positive (78.73%), and 42 are positive for mixed infections (13.33%), which is completely consistent with the positive detection rate of the single PCR detection method.
[Conclusion] This study successfully established a multiplex qRT-PCR method capable of simultaneously detecting six pathogens responsible for calf diarrhea. The method exhibits a detection sensitivity of up to 1 copy/μL for plasmid DNA, shows no cross-reactivity with other common pathogens, and achieves a 100% concordance rate with single PCR method in the detection of 315 clinical samples.
, authors=Guixia ZHU, Shumei YANG, Li ZHONG, Yu WANG, authorsList=Guixia ZHU, Shumei YANG, Li ZHONG, Yu WANG, authorCompany=null, correspAuthors=null, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1297139320062768068, articleId=1297139318028530626, tenantId=1146029695717560320, journalId=1296801528146919473, language=CN, title=多重qRT-PCR检测犊牛腹泻病方法的建立与应用, columnId=1297134043032736530, journalTitle=中国乳业, columnName=疾病防治, runingTitle=null, highlight=null, articleAbstract=
［目的］ 建立一种检测犊牛腹泻病多重qRT-PCR方法，旨在对致犊牛腹泻的多种病原体同时快速检测。
［方法］ 查询多种致犊牛腹泻的病原体保守基因序列作为靶标基因，设计特异性引物和探针，构建质粒标准品，优化引物浓度和退火温度，测试其灵敏度、特异性和临床样本验证。
［结果］ 以103 拷贝/μL质粒作为标准品，成功建立检测犊牛腹泻病多重qRT-PCR方法，反应体系为：10 μL 2×Fast-Start Universal SYBR Green Master，0.5 μL上游引物，0.5 μL下游引物，1 μL模板，8 μL灭菌超纯水，总反应体积为20 μL。反应条件为：95 ℃ 3 min；94 ℃ 20 s、56 ℃ 15 s、72 ℃ 30 s，36 个循环；72 ℃ 5 min。检测犊牛腹泻病多重qRT-PCR方法的灵敏度为1 拷贝/μL，与常见其他病原体无交叉反应，组内和组间变异系数均小于2%。在315 份腹泻犊牛的临床粪便样本中，共检测出248 份阳性（78.73%），42 份为混合感染阳性（13.33%），与单一PCR方法的阳性检出率结果完全一致。
［结论］ 本研究成功建立一种可同时检测6 种犊牛腹泻病原体的多重qRT-PCR方法，对质粒DNA的检测灵敏度可达1 拷贝/μL，与常见其他病原体无交叉反应，与单一PCR方法对315 份临床样本的检测符合率为100%。
, authors=朱贵霞, 杨树美, 钟丽, 王禹, authorsList=朱贵霞, 杨树美, 钟丽, 王禹, authorCompany=null, correspAuthors=null, authorNote=
朱贵霞（1980-），女，山东临沂人，本科，高级兽医师，研究方向为兽医
杨树美（1980-），女，山东临沂人，本科，高级兽医师，研究方向为畜牧兽医
钟丽（1982-），女，贵州贵阳人，硕士，高级畜牧师，研究方向为畜牧
王禹（1975-），男，山东临沂人，本科，中级兽医师，研究方向为兽医。
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发表在《中国乳业》上的论文，视为将本论文的数字化复制权、发行权、汇编权及信息网络传播权许可给《中国乳业》使用。本刊已许可中国学术期刊（光盘版）电子杂志社、北京万方数据股份有限公司等多家单位在其相关数据库产品中以数字化方式复制、汇编、发行、信息网络传播本刊全文。作者向本刊提交文章发表的行为即视为同意我刊授权使用该文著作权。, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=hYWyMEFwldhy5Vk0LllGdQ==, magXml=i6mtf41dPox6HfKXetX50w==, pdfUrl=null, pdf=xyY4jg4SACGv/cPRMHq8AQ==, pdfFileSize=1195210, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=c7KGD8V9AdIIqijEmTWjFw==, mapNumber=null, fund=null)}, authors=[Author(id=1297139320356369353, tenantId=1146029695717560320, journalId=1296801528146919473, articleId=1297139318028530626, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1297139320444449739, tenantId=1146029695717560320, journalId=1296801528146919473, articleId=1297139318028530626, authorId=1297139320356369353, language=EN, stringName=Guixia ZHU, firstName=Guixia, middleName=null, lastName=ZHU, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=Mengyin County Animal Husbandry Development Promotion Center, Linyi Shandong 276200, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1297139320519947212, tenantId=1146029695717560320, journalId=1296801528146919473, articleId=1297139318028530626, authorId=1297139320356369353, language=CN, stringName=朱贵霞, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=山东省临沂市蒙阴县畜牧发展促进中心，山东 临沂 276200, bio={"content":"
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