酶促重组等温扩增实时荧光法快速检测肺炎支原体方法的建立及应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-1529

摘要/Abstract

摘要：

利用酶促重组等温扩增（enzymatic recombinase amplification,ERA）技术建立快速检测肺炎支原体的实时荧光检测方法。针对肺炎支原体P1基因设计特异性引物和探针,优化反应条件,分析其敏感性和特异性,并对临床样本进行验证。ERA实时荧光法在25-40℃均具有扩增能力,在35℃条件下对肺炎支原体的扩增效果最好,20 min内可完成扩增;该法对肺炎支原体的检出限为10³ copies/μL;并对其他6种呼吸道病原体进行检测,均无扩增曲线产生,有较好的特异性;以荧光定量PCR法检测结果为标准,ERA实时荧光法对34份临床样本的检测结果的诊断敏感度为96.15%、特异度为100%、阳性预测值为100%、阴性预测值为88.89%。本研究构建的ERA实时荧光法可以快速简单、灵敏和特异地检测出肺炎支原体,满足现场检测的需求。

关键词: 酶促重组等温扩增技术, 实时荧光检测, 肺炎支原体

Abstract:

This work is to establish a real-time fluorescence detection method of Mycoplasma pneumoniae by the enzymatic recombinase amplification（ERA）technology. Specific primers and probe were designed for M. pneumoniae P1 gene,reaction conditions were optimized,the sensitivity and specificity were evaluated,and the method to clinical samples was validated. The ERA real-time fluorescence method had the amplification ability at 25-40℃,and the amplification effect was the optimal at 35℃,and the amplification was completed within 20 min. The detection limit of M. pneumoniae was 10³ copies/μL. There was no cross reaction with other 6 respiratory pathogens,showing good specificity. The diagnostic sensitivity,specificity,positive predictive value and negative predictive value of ERA real-time fluorescence method for 34 clinical samples were 96.15%,100%,100% and 88.89%,respectively,when the results of quantitative PCR detection were used as the standard. The study established a rapid,sensitive and specific method for early and rapid diagnosis of M. pneumoniae nucleic acid,which meets the needs of on-site rapid detection in primary health institutions.

Key words: enzymatic recombinase amplification（ERA）, real-time fluorescence detection, Mycoplasma pneumoniae

胡海洋, 应婉琴, 何军, 吕芷贤, 谢小平, 邓仲良. 酶促重组等温扩增实时荧光法快速检测肺炎支原体方法的建立及应用[J]. 生物技术通报, 2022, 38(9): 264-270.

HU Hai-yang, YING Wan-qin, HE Jun, LV Zhi-xian, XIE Xiao-ping, DENG Zhong-liang. Establishment and Application of ERA Real-time Fluorescence Method for Rapid Detection of Mycoplasma pneumoniae[J]. Biotechnology Bulletin, 2022, 38(9): 264-270.

图/表 7

表1 ERA实时荧光法引物和探针序列

Table 1 Primer and probe sequences of ERA real-time fluorescence method

名称 Name	序列 Sequence（5'-3'）	长度 Length/bp
Forward Primer 1（F1）	TTGAAGGTGAAGAACGCCG- AGGCGGACAC	29
Forward Primer 2（F2）	CGAAGAGCAATGAAAAACTC- CAGGGCGCTGA	31
Reverse Primer 1（R1）	GGACTGACCCGACTCCTCGCT- CCGCTTAATG	31
Reverse Primer 2（R2）	ATTGGAGTTGCCGCCTGATCC- CGAACTGGAA	31
Probe	TGAAAAAGAAATCGGACTCG- GAGGACAA[FAM-dT]G [THF][BHQ1-dT]CAGCTG- CAGTTAGAA3'Block	46

图1 ERA实时荧光法引物和探针序列

Fig. 1 Primers and probe sequences for ERA real-time fluorescence method

图2 ERA实时荧光法的建立（A）和条件优化（B-C）

Fig. 2 Establishment（A）and condition optimization（B-C）of ERA real-time fluorescence method

图3 ERA实时荧光法敏感性分析

Fig. 3 Sensitivity analysis via ERA real-time fluorescence method

图4 ERA实时荧光法特异性分析

Fig. 4 Specificity analysis via ERA real-time fluorescence method

图5 ERA实时荧光法的临床样本验证

Fig. 5 Clinical sample validation via ERA real-time fluorescence method

表2 ERA实时荧光法与荧光定量PCR法的比较

Table 2 Comparison between ERA real-time fluorescence method and fluorescence qPCR method

	阳性 Positive	阴性 Negative	合计 Total
荧光定量PCR法 Fluorescence qPCR method	26	8	34
ERA实时荧光法 ERA real-time fluorescence method	25	9	34

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