Establishment and Optimization of PCR-ELISA Detection Method for Streptococcus Suis Type 2
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摘要: 根据已发表的猪链球菌2型的cps2j基因序列,设计合成上游标记生物素的1对引物和1条标记地高辛的特异性探针,建立了该菌的PCR-ELISA检测方法。方法利用PCR仪作为杂交场所,并对几个主要条件参数进行优化,结果显示在鲑鱼精DNA浓度1.6mg·mL-1、变性温度为94℃、变性时间为1min、探针浓度为0.5umol·mL-1、杂交温度55℃、杂交时间1min、一抗作用时间45min、二抗作用时间40min时检测方法敏感性最好,特异性最强。通过对17个阴性样本检测,测得平均值为0.057,方差为0.047,计算得临界值为0.198。
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关键词:
- 猪链球菌2型、PCR-ELISA /
- cps2j基因
Abstract: A new PCR-ELISA detection method of Streptococcus suis type 2 has been set up.A pair of primers contained upstream biotin label and a digoxin-marked specific probe were designed according to the published Streptococcus suis type 2 cps2j gene sequences,PCR amplifier was used as the hybrid spaces,and several main parameters were optimized.The results showed that the concentration of salmon sperm DNA of 1.6mg/mL,denaturation temperature of 94℃,denaturation time of 1 min,probe concentration 0.5 μmol·mL-1,hybridization temperature of 55℃,the the hybridization time 1 min,the first anti-role time 45 min,the second anti-role 40 min were the best conditions for sensitivity and specificity.17 negative samples were measured whose average was 0.057,variance was 0.047,and the ritical value was 0.198.-
Key words:
- Streptococcus suis type 2 /
- PCR-ELISA
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[1] STAATS J J,FEDER I,OKWUMABUA O,et al.Streptococcus suis:past and present[J].Veterinary ResCommunications,1997,21:381-407. [2] GOTTSCHALK M,XU J,CALZAS C,et al.Streptococcussuis:a new emerging or an old neglected zoonotic pathogen[J].Future Microbiol,2010,5(3):37-391. [3] 中国兽药典委员会.中华人民共和国兽药典2005年版二部[M].北京:中国农业出版社,2005. [4] 方勤美,黄绍谦,俞伏松,等.猪链球菌2型福建分离株的主要毒力基因分析[J].中国人兽共病患学报,2010,26(1):65-68. [5] 何孔旺,陆承平,倪艳秀.PCR方法检测2型猪链球菌2种毒力因子[J].扬州大学学报,2002,23(3):1-4. [6] SHAMLOUL A M,HADIDI A.Sensitive detection of potatospindle tuber and temperate fruit free viroids by reversetranscription polymerase chain reaction probe capturehybridization[J].J Virol Meth,1999,80:145-155. [7] SMITH H E,DAMMAN M,VAN DER VELDE J,et al.Identification and characterization of the cps locus ofStreptococcus suis sero-type 2:the capsuLe protects againstphagocytosis and is an important viruLence factor[J].InfectImmun,1999,67(4):1750-1756. -
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