中国奶牛
中國奶牛
중국내우
CHINA DAIRY CATTLE
2012年
21期
4-9
,共6页
肖淦文%陈颖钰%彭清洁%胡长敏%崔朋%巴晓亮%陈焕春%郭爱珍
肖淦文%陳穎鈺%彭清潔%鬍長敏%崔朋%巴曉亮%陳煥春%郭愛珍
초감문%진영옥%팽청길%호장민%최붕%파효량%진환춘%곽애진
多重PCR%牛支原体%多杀性巴氏杆菌A型%化脓隐秘杆菌%牛呼吸疾病综合征
多重PCR%牛支原體%多殺性巴氏桿菌A型%化膿隱祕桿菌%牛呼吸疾病綜閤徵
다중PCR%우지원체%다살성파씨간균A형%화농은비간균%우호흡질병종합정
Multiple PCR%Mycoplasma bovis%Pasteurella multocida type A%Arcanobacterium pyogenes%Bovinerespiratory disease complex
本试验旨在建立一种可同时鉴别牛支原体、巴氏杆菌A型和化脓隐秘杆菌的多重PCR方法。分别针对多杀性巴氏杆菌A型特异的hyac-hvaD基因区段、化脓隐秘杆菌的16SrRNA基因上保守区段和牛支原体的UvrC基因设计特异性引物，多重PCR的最佳扩增条件确定为：95℃ 10min预变性；95℃ 1min，56℃ 50s，72℃ 1min，循环30次；72℃ 210min延伸。结果表明，该多重PCR方法可同时扩增出以上三种致病菌的特异性片段，不能扩增出其他病原菌的相关片段；对多杀性巴氏杆菌A型、化脓隐秘杆菌和牛支原体的最低检测浓度分别为8×10^5CFU／mL、8×10^5CFU／mL和4×10^6CFU／mL。同时用该方法检测了牛支原体肺炎患牛的鼻拭子与肺组织，发现12h预增菌后，肺组织检测与牛支原体培养的阳性符合率为92％。对临床样本进行牛支原体分离培养需要3-4d时间，而采用多重PCR方法检测12h预增菌则能在24h内出结果。该多重PCR方法显著加快了临床诊断速度，具有推广应用价值。
本試驗旨在建立一種可同時鑒彆牛支原體、巴氏桿菌A型和化膿隱祕桿菌的多重PCR方法。分彆針對多殺性巴氏桿菌A型特異的hyac-hvaD基因區段、化膿隱祕桿菌的16SrRNA基因上保守區段和牛支原體的UvrC基因設計特異性引物，多重PCR的最佳擴增條件確定為：95℃ 10min預變性；95℃ 1min，56℃ 50s，72℃ 1min，循環30次；72℃ 210min延伸。結果錶明，該多重PCR方法可同時擴增齣以上三種緻病菌的特異性片段，不能擴增齣其他病原菌的相關片段；對多殺性巴氏桿菌A型、化膿隱祕桿菌和牛支原體的最低檢測濃度分彆為8×10^5CFU／mL、8×10^5CFU／mL和4×10^6CFU／mL。同時用該方法檢測瞭牛支原體肺炎患牛的鼻拭子與肺組織，髮現12h預增菌後，肺組織檢測與牛支原體培養的暘性符閤率為92％。對臨床樣本進行牛支原體分離培養需要3-4d時間，而採用多重PCR方法檢測12h預增菌則能在24h內齣結果。該多重PCR方法顯著加快瞭臨床診斷速度，具有推廣應用價值。
본시험지재건립일충가동시감별우지원체、파씨간균A형화화농은비간균적다중PCR방법。분별침대다살성파씨간균A형특이적hyac-hvaD기인구단、화농은비간균적16SrRNA기인상보수구단화우지원체적UvrC기인설계특이성인물，다중PCR적최가확증조건학정위：95℃ 10min예변성；95℃ 1min，56℃ 50s，72℃ 1min，순배30차；72℃ 210min연신。결과표명，해다중PCR방법가동시확증출이상삼충치병균적특이성편단，불능확증출기타병원균적상관편단；대다살성파씨간균A형、화농은비간균화우지원체적최저검측농도분별위8×10^5CFU／mL、8×10^5CFU／mL화4×10^6CFU／mL。동시용해방법검측료우지원체폐염환우적비식자여폐조직，발현12h예증균후，폐조직검측여우지원체배양적양성부합솔위92％。대림상양본진행우지원체분리배양수요3-4d시간，이채용다중PCR방법검측12h예증균칙능재24h내출결과。해다중PCR방법현저가쾌료림상진단속도，구유추엄응용개치。
A triple-PCR method was developed to detected Mycoplasma bovis, Pasteurella multocida type A, and Arcanobacterium pyogenes simutanously. The primer sets were designed specifically to hyaC--hyaD gene segment ofP. multocida type A, conservative section of 16S rRNA gene ofA. pyogenes and the UvrC gene of M. bovis and screened. Finally, one set of primers for each pathogen was selected, and the condition of the multiple PCR was optimized as follows: 95℃ 10 rain, 95℃ lmin, 56℃ 50s, 72℃ lmin for 30 cycles, 72℃ 10min for extension. The specificity tests showed that this multiple PCR method amplified all three target gene fragments from these three pathogens, and did not yield any products for the DNA templates from other common bacterial pathogens of cattle. Sensitivity tests showed that the sensitivities of this multiple PCR for P, multocida type A and A. pyogenes is 8×10^5CFU/mL, and 4×10^6CFU/mL for M. bovis. The detection of M. bovis in lesioned lung and nasal swabs from diseased beef cattle with M. boris pneumonia with this triple-PCR produced a 92% positive agreement with M. bovis culture for the 12h pre-culture of lung samples. Compared to the time of 3-4 d required by 34.. bovis culture, this multiple PCR combined 12h pre-culture can develop the results within 24h and shows a promising application in clinical diagnosis.
