| 摘要: |
| 【目的】建立一种可快速检测副猪嗜血杆菌的环介导等温扩增(LAMP)方法。【方法】根据GenBank中副猪嗜血杆菌肽聚糖相关脂蛋白(PalA)基因序列，在其保守区域设计外引物、内引物和环引物用于LAMP检测。优化副猪嗜血杆菌LAMP检测的反应体系，在反应产物中加入SYBR Green Ⅰ，对检测结果进行肉眼判定，建立副猪嗜血杆菌可视化LAMP检测方法，评价该检测方法的特异性、敏感性。用建立的副猪嗜血杆菌可视化LAMP检测方法对临床分离的8株不同血清型副猪嗜血杆菌进行检测。从疑似患副猪嗜血杆菌病的猪体内采集10种体液，用所建立的可视化LAMP检测方法进行检测。【结果】建立了副猪嗜血杆菌可视化LAMP检测方法，该法在55 ℃水浴1 h即可对副猪嗜血杆菌核酸进行高效扩增，反应结束后加入SYBR Green Ⅰ即可通过肉眼观察对结果进行判断。该方法具有很强的特异性，其对DNA核酸的最低检测限为40 fg，是常规PCR检测最低限的100倍，显示出较高的敏感性；用建立的LAMP方法对8株不同血清型副猪嗜血杆菌进行检测，结果均为阳性。用建立的LAMP方法对10种体液进行检测，结果鼻液、气管液、胸腔渗出物、心脏血、脑膜液、腹腔液和关节囊液呈阳性，唾液、心包液和尿液呈阴性。【结论】建立了一种可对副猪嗜血杆菌进行快速检测并可凭肉眼判定结果的可视化LAMP方法，该法操作简便、反应快速、敏感性强、特异性高，适合在兽医基层进行推广应用。 |
| 关键词: 副猪嗜血杆菌 肽聚糖相关脂蛋白 环介导等温扩增技术 |
| DOI： |
| 分类号: |
| 基金项目:福建省公益类科研专项（2010R1025 2）；国家公益性行业（农业）科研专项（NYHYZX07-034）；福建省农业科学院创新团队项目(CXTD-1-1306) |
|
| Development and application of a loop-mediated isothermal amplification assay for visual detection of Haemophilus parasuis |
|
CHE Yongliang, CHEN Rujing, WANG Longbai,et al
|
|
Fujian Academy of Agricultural Sciences
|
| Abstract: |
| 【Objective】A rapid detection method for Haemophilus parasuis (Hps) by loop-mediated isothermal amplification assay (LAMP) was established in this study.【Method】According to the published peptidoglycan-associated lipoprotein (PalA) gene sequences of Hps in GenBank,targeting the conserved region of palA gene,3 pairs of primers (F3 and B3,BIP and FIP,and LF and LB) were designed to establish a optimized LAMP system which can detect Hps.The specificity and sensitivity of the LAMP method were evaluated.Meanwhile,the amplified products were colored by SYBR Green I after completion of the reaction,so that the amplification can be observed with naked eyes.8 serotypic Hps strains were detected with the LAMP method.10 kinds of body fluids were collected from pigs suspected of suffering from Hps,and the distribution of Hps in vivo was analyzed by the established LAMP method.【Result】LAMP efficiently amplified Hps genomic DNA in 55 ℃ water for 1 h.The results could be detected with naked eyes after addition of SYBR Green I.The LAMP had a strong specificity with a detection limit of 40 fg DNA,which was 100 times higher than that of PCR.The detected results of 8 serotypic Hps strains with LAMP method were consistent with PCR.Among the 10 types of collected body fluids,Hps was detected in nasal drops,tracheal fluid,pleural exudates,heart blood,meningeal liquid,peritoneal fluid and articular capsule liquid,while it was not found in saliva,pericardial fluid and urine by the LAMP method.【Conclusion】A visualized LAMP method with easy operation,fast detection,high sensitivity and specificity to detect Hps was established,and the method was suitable for large scale application in grass roots. |
| Key words: Haemophilus parasuis peptidoglycan-associated lipoprotein(PalA) loop-mediated isothermal amplification(LAMP) |
