茄科雷尔氏菌IMSA-LAMP检测方法的建立

袁婷; 罗龙辉; 张雪吟; 刘吉平

doi:10.7671/j.issn.1001-411X.202203030

茄科雷尔氏菌IMSA-LAMP检测方法的建立

Establishment of IMSA-LAMP detection method for Ralstonia solanacearu

摘要

摘要:
目的桑树青枯病是由茄科雷尔氏菌Ralstonia solanacearum引起的一种危害严重的细菌性病害，建立一种快速、灵敏的茄科雷尔氏菌检测方法，对桑树青枯病的有效控制有重要意义。

方法本研究以茄科雷尔氏菌果胶裂解酶基因(Pectate lyase gene)为靶标，基于等温多自配引发扩增技术(Isothermal multiple self-matching-initiated amplification，IMSA)的引物设计原理，结合环介导等温扩增技术(Loop-mediated isothermal amplification，LAMP)的反应体系，建立一种快速有效检测茄科雷尔氏菌的IMSA-LAMP法，并对该方法的最佳反应参数进行了筛选。

结果基于果胶裂解酶基因建立的IMSA-LAMP检测方法在64.5 ℃条件下，45 min内可完成对阳性样品的特异检测，对茄科雷尔氏菌的模板DNA检测灵敏度达200 fg/µL (对应菌为1×10² CFU/mL)；对生产上收集的疑似桑树青枯病病样的检出率为87.5%。

结论 IMSA-LAMP检测方法具有良好的实用性，可为桑树青枯病的快速检测、诊断与防疫提供新的技术支持。

Abstract:
Objective Mulberry bacterial wilt is a seriously harmful bacterial disease caused by Ralstonia solanacearum. Therefore, it is of great significance to establish a rapid and sensitive detection method for R. solanacearum to effectively control mulberry bacterial wilt.

Method Pectate lyase gene of R. solanacearum was used as the target. Based on the primer design principle of isothermal multiple self-matching-initiated amplification (IMSA), combining the loop-mediated isothermal amplification (LAMP) reaction system, a rapid and effective IMSA-LAMP method for detection ofR. solanacearum was established. The optimal reaction parameters of this method were screened.

Result The IMSA-LAMP method based on pectate lyase gene could complete the specific detection of positive samples within 45 min at 64.5 ℃, and the detection sensitivity of R. solanacearum template DNA was 200 fg/μL (the corresponding bacteria detection sensitivity was 1 × 10² CFU/mL); The detection rate of suspected mulberry bacterial wilt samples collected in production was 87.5%.

Conclusion This method has good practicability and can provide new technical support for the rapid detection, diagnosis and epidemic prevention of mulberry bacterial wilt.

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