鸚鵡熱衣原體（Chlamydia psittaci）是一種人獸共患病原體，能引起自然疫源性衣原體病，目前廣泛分布于世界各地。本研究應(yīng)用環(huán)介導(dǎo)等溫?cái)U(kuò)增技術(shù)（LAMP），針對鸚鵡熱衣原體23S RNA基因序列設(shè)計(jì)引物，并進(jìn)行篩選以及敏感性和特異性試驗(yàn)，建立了鸚鵡熱衣原體恒溫?zé)晒鈹U(kuò)增方法。該方法在63 ℃恒溫條件下1 h內(nèi)即可顯示結(jié)果，操作簡單、快速，特異性強(qiáng)，靈敏度可達(dá)100拷貝/μL，且與流產(chǎn)衣原體、動(dòng)物布魯氏菌、牛結(jié)核分枝桿菌、沙門氏菌、大腸桿菌、金黃色葡萄球菌無交叉反應(yīng)；用國產(chǎn)儀器可直接通過檢測熒光信號判讀結(jié)果，既增強(qiáng)了準(zhǔn)確性，也避免了開蓋污染產(chǎn)生假陽性；應(yīng)用建立的LAMP方法對實(shí)驗(yàn)室保存的臨床DNA樣品進(jìn)行檢測，發(fā)現(xiàn)檢測結(jié)果與QPCR相同。該方法的建立彌補(bǔ)了傳統(tǒng)檢測技術(shù)的不足，為實(shí)現(xiàn)鸚鵡熱衣原體的現(xiàn)場快速診斷提供了技術(shù)支持。
Establishment of a Loop-mediated Isothermal Amplification Method for Chlamydia psittaci
Chlamydia psittaci，a kind of zoonotic pathogen，is widely distributed all over the world，by which，chlamydiosis of natural origin might be caused. In this study，the primers were designed based on 23S RNA gene sequence of Chlamydia psittaci，and then screened. After sensitivity and specificity tests，a thermostatic fluorescence amplification method for Chlamydia psittaci was established according to the loop-mediated isothermal amplification method（LAMP）. By the established method，test results could be shown within one hour under the constant temperature of 63 ℃ due to its advantages including simple operation，rapidity，strong specificity，sensitivity of 100 copies/μL and no cross-reaction with Chlamydia abortus，Brucella animalis，Mycobacterium bovis，Salmonella，Escherichia coli and Staphylococcus aureus. The results could be interpreted directly by fluorescence signals with domestic instruments，which not only enhanced the accuracy，but also avoided false positive due to pollution brought by cap opening. The clinical DNA samples stored in laboratories were detected by the LAMP method，it was found that the results were same as that by QPCR. It was concluded that traditional methods were remedied by the established method，which provided technical support for rapid field detection of Chlamydia psittaci.
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