目的 探讨基于特异常规引物（即引物不需折叠成特定二级结构）靶向恒温扩增重复DNA序列的方法，并测试其检测结核分枝杆菌（Mycobacterium tuberculosis，Mtb）的可能性。方法 以合成的重复DNA或以抽提的细菌基因组DNA为模板，用Bst 2.0 WarmStart DNA聚合酶进行恒温扩增。用琼脂糖凝胶电泳检测扩增结果。结果 合成的重复DNA可用其特异常规引物进行恒温扩增。进一步，Mtb H37Rv Ⅱ型分枝杆菌散在重复单位（mycobacterial interspersed repetitive units，MIRUs）可用其特异常规引物对（即Ⅱ_MIRU-F和Ⅱ_MIRU-R）或单引物（即Ⅱ_MIRU-F）进行恒温扩增。相比于非分枝杆菌菌株、非结核分枝杆菌菌株、Mtb复合物菌株和临床分离株，Ⅱ_MIRU-F能够高特异地扩增Mtb菌株。Ⅱ_MIRU-F针对Mtb H37Rv基因组DNA的检测下限较高，且不能特异地区分Mtb阴性痰液样本和Mtb阳性痰液样本。结论 常规引物可恒温扩增重复DNA序列（包括Mtb Ⅱ型MIRUs）；Mtb Ⅱ型MIRUs特异引物Ⅱ_MIRU-F不适合用于痰液样本的检测。
Abstract
Objective To investigate the isothermal amplification approach targeting repetitive DNA sequences using ordinary primers being not able to fold into designated secondary structures.Then the novel approach was utilized to detect Mycobacterium tuberculosis (Mtb). Methods Isothermal amplification was applied using synthesized repetitive DNA sequence or extracted bacteria genomic DNA as templates,and using Bst 2.0 WarmStart DNA as DNA polymerase.The DNA products were detected via agarose gel electrophoresis. Results Synthesized repetitive DNA sequence could be isothermally amplified using its sequence-specific ordinary primers.Subsequently,primers (i.e.Ⅱ_MIRU-F/Ⅱ_MIRU-R) targeting type Ⅱ mycobacterial interspersed repetitive units (MIRUs) of Mtb H37Rv were designed.Primer pair Ⅱ_MIRU-F/Ⅱ_MIRU-R or single primer Ⅱ_MIRU-F could amplify Mtb H37Rv genomic DNA isothermally.Ⅱ_MIRU-F could amplify Mtb with high specificity,after comparing its performances among non-mycobacteria strains,non-tuberculous mycobacteria strains,Mtb complex strains,and Mtb clinical isolates.Ⅱ_MIRU-F had high limit of detection against Mtb H37Rv genomic DNA,and couldn't specifically distinguish Mtb-positive sputum samples and Mtb-negative sputum samples. Conclusion Repetitive DNA sequences (including Mtb type Ⅱ MIRUs) could be isothermally amplified;Ⅱ_MIRU-F (specific to Mtb type Ⅱ MIRUs) does not suit to be utilized to test sputum samples.
关键词
恒温扩增 /
重复DNA /
结核分枝杆菌 /
Ⅱ型分枝杆菌散在重复单位
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Key words
isothermal amplification /
repetitive DNA /
Mycobacterium tuberculosis /
type Ⅱ mycobacterial interspersed repetitive units
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中图分类号：
R378.91+1
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脚注
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基金
国家重点研发计划（2018YFD0500900）；国家科技重大专项（2018ZX10731301，2018ZX10302301）；上海市科委医学引导项目（18411970700）
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