中国动物检疫
中國動物檢疫
중국동물검역
CHINA ANMAL QUARANTINE
2014年
10期
57-61,65
,共6页
王彩霞%林祥梅%张永宁%吕继洲%冯春燕%袁向芬%邓俊花%吴绍强
王綵霞%林祥梅%張永寧%呂繼洲%馮春燕%袁嚮芬%鄧俊花%吳紹彊
왕채하%림상매%장영저%려계주%풍춘연%원향분%산준화%오소강
施马伦贝格病毒%聚合酶链反应%焦磷酸测序%鉴定%检测
施馬倫貝格病毒%聚閤酶鏈反應%焦燐痠測序%鑒定%檢測
시마륜패격병독%취합매련반응%초린산측서%감정%검측
Schmallenberg disease%polymerase chain reaction%pyrosequencing%identification%detection
为适应口岸施马伦贝格病快速、准确、高通量检测需求,建立一种基于RT-PCR及焦磷酸测序技术平台的施马伦贝格病检测方法。本研究针对施马伦贝格病毒基因组RNA的S、M和L三个基因节段保守区域利用焦磷酸测序软件PyroMark Q96ID分别设计RT-PCR扩增引物及焦磷酸测序引物,以人工合成的SBV重组质粒为模板分别建立了SBV S、M、L基因的PCR-焦磷酸测序检测方法,比较三基因的PCR扩增结果以及测序分析结果,结合特异性检测结果,最终确定以M基因作为靶基因建立的焦磷酸测序检测方法效果最好,并以德国FLI提供的SBV(BH80株)RNA对所建立的方法进行验证,通过对测得序列的比对分析可确定为施马伦贝格病毒,这表明本研究所建立的方法灵敏度高、稳定性好、特异性强,可以用于施马伦贝格病毒基因序列水平上的精准检测鉴定。
為適應口岸施馬倫貝格病快速、準確、高通量檢測需求,建立一種基于RT-PCR及焦燐痠測序技術平檯的施馬倫貝格病檢測方法。本研究針對施馬倫貝格病毒基因組RNA的S、M和L三箇基因節段保守區域利用焦燐痠測序軟件PyroMark Q96ID分彆設計RT-PCR擴增引物及焦燐痠測序引物,以人工閤成的SBV重組質粒為模闆分彆建立瞭SBV S、M、L基因的PCR-焦燐痠測序檢測方法,比較三基因的PCR擴增結果以及測序分析結果,結閤特異性檢測結果,最終確定以M基因作為靶基因建立的焦燐痠測序檢測方法效果最好,併以德國FLI提供的SBV(BH80株)RNA對所建立的方法進行驗證,通過對測得序列的比對分析可確定為施馬倫貝格病毒,這錶明本研究所建立的方法靈敏度高、穩定性好、特異性彊,可以用于施馬倫貝格病毒基因序列水平上的精準檢測鑒定。
위괄응구안시마륜패격병쾌속、준학、고통량검측수구,건립일충기우RT-PCR급초린산측서기술평태적시마륜패격병검측방법。본연구침대시마륜패격병독기인조RNA적S、M화L삼개기인절단보수구역이용초린산측서연건PyroMark Q96ID분별설계RT-PCR확증인물급초린산측서인물,이인공합성적SBV중조질립위모판분별건립료SBV S、M、L기인적PCR-초린산측서검측방법,비교삼기인적PCR확증결과이급측서분석결과,결합특이성검측결과,최종학정이M기인작위파기인건립적초린산측서검측방법효과최호,병이덕국FLI제공적SBV(BH80주)RNA대소건립적방법진행험증,통과대측득서렬적비대분석가학정위시마륜패격병독,저표명본연구소건립적방법령민도고、은정성호、특이성강,가이용우시마륜패격병독기인서렬수평상적정준검측감정。
To meet the rapid,accurate and high throughput detection of Schmallenberg disease,a pyrosequencing assay was developed in combination with PCR amplification of the target sequences. Pyrosequencing specific primers of SBV segments of S,M and L were separately designed targeting the conserved region of the sequence using primer designer software PyroMark Q96ID. PCR-pyrosequencing assay of SBV S,M,L were established with the recombi-nant plasmid of SBV S,M,L as template,and the sequences were determined by PyroMarkTM ID System. SBV RNA presented by FLI was used as positive material to validate the developed PCR-pyrosequencing assay. Comparing the electrophoresis result of PCR amplicons,the result of pyrosequencing assay and the result of specificity detection,the pyrosequencing assay targeting M gene was the best one among the assays targeting S,M and L genes. The developed SBV-M pyrosequencing assay was further verified by using the SBV positive RNA material as template by means of Blast analysis of obtained sequence online. Results showed that the developed pyrosequencing assay could identify SBV. The method was of high sensitivity,good stability and high specificity,and could be used as an accurate detection method for Schmallenberg disease .