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首页> 外文期刊>Tropical Animal Health and Production >Comparative diagnostic evaluation of OMP31 gene based TaqManA (R) real-time PCR assay with visual LAMP assay and indirect ELISA for caprine brucellosis
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Comparative diagnostic evaluation of OMP31 gene based TaqManA (R) real-time PCR assay with visual LAMP assay and indirect ELISA for caprine brucellosis

机译：基于Taqmana（R）实时PCR测定与可视灯测定的比较诊断评价和Caprine Brucellosis的间接ELISA

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Brucellosis is one of the leading causes of abortion in domestic animals that imposes costs on both economy and society. The disease is highly zoonotic and poses risk to animal handlers due to its zoonotic nature. It causes stillbirth, loss of kids and abortion in last term of pregnancy. Reproductive damage includes infertility in does and orchitis and epididymitis in breeding bucks, which result in high financial losses to farmers and the agriculture industry as a whole. It requires highly sensitive and specific assays to diagnose the disease at field level. In the current study, a visual loop-mediated isothermal amplification (LAMP) assay and the TaqManA (R) real-time PCR were developed with high sensitivity and specificity. For the TaqManA (R) probe, real-time PCR primers were developed using Omp31 gene as target and primers were designed using discontiguous conserved sequences of Omp31 gene. The Omp31 probes were designed by attaching 6-FAM reporter dye at the 5' end and BHQ-1 quencher at the 3' end. Published primers were used for visual LAMP assay targeting the Omp25 gene. Sensitivity of the standardized visual LAMP assay and TaqManA (R) real-time PCR assay was determined by serial dilution of positive Brucella melitensis DNA (10(2) to 10(-4) ng) obtained from standard culture. The TaqManA (R) probe real-time assay can detect as low as 100 fg of B. melitensis DNA, whereas culture from vaginal swab washings has a limit of detection (LOD) of only 1 cfu/ml. Similarly, the visual LAMP assay can detect as low as 10 fg of B. melitensis DNA as compared to an LOD of 30 cfu/ml from culture of vaginal swab washings. Both assays were compared with serological tests (serum tube agglutination test (STAT) and indirect enzyme-linked immunosorbent assay (iELISA)) for diagnostic sensitivity and specificity. Diagnostic sensitivities and specificities for TaqManA (R) real-time PCR vs. LAMP assays were 98 and 100% vs. 100 and 97.8%, respectively. Results of visual LAMP assay indicated that LAMP is a fast, specific, sensitive, inexpensive and suitable method for diagnosis of B. melitensis infection under field conditions. On the other hand, Omp31 TaqManA (R) probe real-time assay can be used in conjunction with the other field-based diagnostic tests due to its high specificity.
机译：布鲁氏菌病是导致家畜流产的主要原因之一，对经济和社会都造成了损失。这种疾病具有高度的人畜共患性，由于其人畜共患性，给动物饲养者带来风险。它会导致死产、失去孩子和最后一个妊娠期流产。生殖损害包括母鹿的不孕和繁殖雄鹿的睾丸炎和附睾炎，这给农民和整个农业造成了巨大的经济损失。它需要高灵敏度和特异性的检测来在现场水平诊断该疾病。在目前的研究中，开发了一种具有高灵敏度和特异性的视觉环介导等温扩增（LAMP）分析和TaqManA（R）实时PCR。对于TaqManA（R）探针，以Omp31基因为靶点开发实时PCR引物，并使用Omp31基因的不连续保守序列设计引物。Omp31探针的设计方法是在5'端连接6-FAM报告染料，在3'端连接BHQ-1猝灭剂。已发表的引物用于靶向Omp25基因的目测LAMP分析。通过从标准培养物中获得的阳性布鲁氏菌DNA（10（2）至10（-4）ng）的连续稀释，测定标准化目视灯法和TaqManA（R）实时PCR法的灵敏度。TaqManA（R）探针实时检测法可检测到低至100 fg的梅里滕斯双歧杆菌DNA，而阴道拭子洗涤液培养物的检测限（LOD）仅为1 cfu/ml。同样，与阴道拭子洗涤液培养物的LOD为30 cfu/ml相比，目视灯检测法可检测到低至10 fg的梅里滕斯双歧杆菌DNA。在诊断敏感性和特异性方面，将两种检测方法与血清学试验（血清管凝集试验（STAT）和间接酶联免疫吸附试验（iELISA））进行比较。TaqManA（R）实时PCR与LAMP检测的诊断敏感性和特异性分别为98%和100%，而LAMP检测的诊断敏感性和特异性分别为100%和97.8%。结果表明，LAMP是一种快速、特异、灵敏、廉价、适用于田间条件下梅毒双歧杆菌感染诊断的方法。另一方面，Omp31 TaqManA（R）探针实时分析由于其高特异性，可与其他基于现场的诊断试验结合使用。

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