Establishment and Application of ERA Real-time Fluorescence Method for Rapid Detection of Mycoplasma pneumoniae

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

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

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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