【摘要】：采用環(huán)介導(dǎo)等溫擴增(LAMP)技術(shù),以豬鼻支原體的P37基因序列為靶基因,設(shè)計1組特異性引物,對反應(yīng)體系的引物濃度、MgSO_4、dNTPs、反應(yīng)溫度和時間進行優(yōu)化,并進行了特異性和靈敏度試驗,旨在建立豬鼻支原體(Mhr)的簡便、快速、準確的分子檢測方法。同時應(yīng)用所建立的方法對臨床樣品進行檢測,結(jié)果顯示,最佳反應(yīng)體系為MgSO_46 mmol/L,dNTPs 1.2 mmol/L,內(nèi)引物(FIP/BIP)1.6 μmol/L,外引物(F3/B3)0.2 μmol/L,Bst DNA聚合酶8 U,60℃擴增60 min。靈敏度是常規(guī)PCR的100倍并且與其他病原菌無任何交叉反應(yīng)。對23份呼吸道疾病臨床樣品進行檢測的結(jié)果顯示,普通PCR和LAMP的檢出率分別為23.4%和79.6%。表明建立的LAMP檢測方法可以用于Mhr的常規(guī)檢疫。
[Abstract]:A group of specific primers were designed using the sequence of P37 gene of Mycoplasma suis as target gene. The concentration of primers, the temperature and time of MgSO_4,dNTPs, reaction were optimized. The specificity and sensitivity tests were carried out to establish a simple, rapid and accurate molecular detection method for Mycoplasma suis (Mhr). The results showed that the best reaction system was MgSO_46 mmol/L,dNTPs 1.2 mmol/L, internal primer (FIP/BIP) 1.6 渭 mol/L, external primer (F3/B3) 0.2 渭 mol/L,. Amplification of Bst DNA Polymerase 8U at 60 鈩，

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