CAJ | 학술논문

针对副猪嗜血杆菌（Hps）16 S rRNA 序列和猪传染性胸膜放线杆菌（App）ApxIVA 基因序列各设计一对引物，分别能扩增大小为822 bp 和346 bp 的目的基因片段，建立快速鉴定 Hps 和 App 的双重 PCR 方法。特异性试验表明，该方法对波氏杆菌、巴氏杆菌、大肠埃希菌、沙门菌、金黄色葡萄球菌、猪圆环病毒2型、猪流感病毒、猪繁殖与呼吸综合征病毒的扩增结果均为阴性。敏感性试验表明，该方法与单项 PCR 的敏感性一致，最低可检测核酸浓度 Hps 38 pg/mL，App 3．8 pg/mL；最低检测细菌含量 Hps 8．9 cfu，App 26．9 cfu。利用该方法检测 Hps 和 App 参考菌株、临床分离株、36份临床肺组织样本和人工感染的猪肺组织样本。结果显示，该方法对参考菌株和临床分离株全部检出，36份临床样本中检出 Hps 18份，App 为阴性，与单项 PCR 检测结果一致，人工感染组织样本也均能检出。表明该方法适用于 Hps 和 App 的临床快速检测。
침대부저기혈간균（Hps）16 S rRNA 서렬화저전염성흉막방선간균（App）ApxIVA 기인서렬각설계일대인물，분별능확증대소위822 bp 화346 bp 적목적기인편단，건립쾌속감정 Hps 화 App 적쌍중 PCR 방법。특이성시험표명，해방법대파씨간균、파씨간균、대장애희균、사문균、금황색포도구균、저원배병독2형、저류감병독、저번식여호흡종합정병독적확증결과균위음성。민감성시험표명，해방법여단항 PCR 적민감성일치，최저가검측핵산농도 Hps 38 pg/mL，App 3．8 pg/mL；최저검측세균함량 Hps 8．9 cfu，App 26．9 cfu。이용해방법검측 Hps 화 App 삼고균주、림상분리주、36빈림상폐조직양본화인공감염적저폐조직양본。결과현시，해방법대삼고균주화림상분리주전부검출，36빈림상양본중검출 Hps 18빈，App 위음성，여단항 PCR 검측결과일치，인공감염조직양본야균능검출。표명해방법괄용우 Hps 화 App 적림상쾌속검측。
The duplex PCR was developed for detecting Haemophilus parasuis (Hps)and Actinobacillus pleuropneumoniae (App).The 821 bp and 346 bp DNA fragments were specifically amplified,respective-ly,which were based on 16 S rRNA of Hps gene and App ApxIVA.The assay could not amplify any genes of Bordetella bronchiseptica ,Pasteurella multocida ,Escherichia coli ,Salmonella,Staphylococcus au-reus ,PCV-2,SIV and PRRSV.The duplex PCR could detect 38 pg/mL of Hps,3.8 pg/mL of App for DNA,as well as 8.9 cfu of Hps and 26.9 cfu of App for bacteria.The duplex PCR and bacterial culture were used to analyze lung samples from slaughter house pigs.The duplex PCR was positive in 18 of 36 clinical samples and Hps was isolated from 13 of 36 of these samples,which were consistent with the sin-gle PCR method to detect Hps and App.Furthermore,all of reference and clinical strains and artificial samples could be identified by this method.Therefore,the results showed that the duplex PCR is suitable for the detection of Hps and App in clinic.