Establishment of Dual LFD-RPA Rapid Detection Method for Mycoplasma gallisepticum and Mycoplasma synoviae

doi:10.13560/j.cnki.biotech.bull.1985.2023-1107

Abstract

Abstract:

【Objective】 Mycoplasma gallisepticum（MG）and Mycoplasma synoviae（MS）are the two most harmful mycoplasmas to poultry. Early diagnosis of MG and MS was a key to prevent and control avian mycoplasma. Through combining recombinase polymerase amplification（RPA）technology with lateral flow dipstick（LFD）, a rapid detection method for MG and MS with convenient operation, rapid response and visual results was established. 【Method】 Specific primers and probes were designed based on MG mgc2 gene and MS vlhA gene, and a dual LFD-RPA rapid detection method was established by optimizing reaction time, reaction temperature and primer ratio, and its sensitivity, specificity and stability were evaluated. Meanwhile, 120 clinical samples were tested. 【Result】 The amplification was done by dual LFD-RPA detection method at 37℃ for 5-10 min, and the optimal primer ratio of RPA-MG-F2/R2 and RPA-MS-F1/R1 was 0.6∶1.4. The minimum detection limits of MG and MS were 3.75 copies/μL and 3.46 copies/μL, respectively. This method and the PCR method recommended by OIE were used to detect 120 samples, and the coincidence rate was 93.3%. 【Conclusion】 The amplification can be done by the dual LFD-RPA detection method of MG and MS under constant temperature conditions. It has simple operation, rapid response, high sensitivity, strong specificity and good stability, and the results can be observed without using any instrument. It is suitable for the field rapid detection of MG and MS infection alone or mixed infection at the primary level.

Key words: Mycoplasma gallisepticum, Mycoplasma synoviae, recombinase polymerase amplification, lateral flow dipstick

TIAN Xing-miao, WANG Jian-lin, GUO Lei, SI Duo-duo, GONG Zhen-xing, LI Ji-dong. Establishment of Dual LFD-RPA Rapid Detection Method for Mycoplasma gallisepticum and Mycoplasma synoviae[J]. Biotechnology Bulletin, 2024, 40(7): 117-124.

Figures/Tables 12

References 21

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引物名称 Primer name	引物序列 Primer sequence（5'-3'）	片段大小 Product length/bp
PCR-MG-F	GTGTTCTCGGGTGCTATTGCTC	819
PCR-MG-R	TGGGGGCTGATTCATTCCTTGA	819
PCR-MS-F	TAGACCCTTAATCGAATTTTG	506
PCR-MS-R	CTTTTGAGTATTCAGCATCTTC	506

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	片段大小 Product length/bp
PCR-MG-F	GTGTTCTCGGGTGCTATTGCTC	819
PCR-MG-R	TGGGGGCTGATTCATTCCTTGA	819
PCR-MS-F	TAGACCCTTAATCGAATTTTG	506
PCR-MS-R	CTTTTGAGTATTCAGCATCTTC	506

引物名称Primer name	引物序列Primer sequence（5'-3'）	片段大小Product length/bp
RPA-MG-F1	AGATTACCTCCGAACCCTGATTTTATCCAGT	362
RPA-MG-R1	AATTCTTTGTTGGGGATTAGGACCAAATTGC	362
RPA-MG-F2	CCTCCGAACCCTGATTTTATCCAGTAGTG	202
RPA-MG-R2	GTTGGCTCAATCGCTTCTGTTTTATTTTGT	202
RPA-MG-F3	ATTTGGTCCTAATCCCCAACAAAGAATTAACCC	301
RPA-MG-R3	ATTAAACCCACCTCCAGCTTTATTTCCCATCG	301
RPA-MS-F1	AAAATTAAATTACTATTAGCAGCTAGTGCAGT	130
RPA-MS-R1	GGTTTTGAGGATTATCAGTATTTGGGTTTCCAG	130
RPA-MS-F2	AATTAAATTACTATTAGCAGCTAGTGCAGT	244
RPA-MS-R2	TTCTGTTTTAGCAGCCTCTACAGGGTCAAC	244
RPA-MS-F3	CCTGAACCAACACCTGGAAACCCAAATACTG	167
RPA-MS-R3	TTAGCTTCTGTTTTAGCAGCCTCTACAGGGTC	167

引物名称Primer name	引物序列Primer sequence（5'-3'）	片段大小Product length/bp
RPA-MG-F1	AGATTACCTCCGAACCCTGATTTTATCCAGT	362
RPA-MG-R1	AATTCTTTGTTGGGGATTAGGACCAAATTGC	362
RPA-MG-F2	CCTCCGAACCCTGATTTTATCCAGTAGTG	202
RPA-MG-R2	GTTGGCTCAATCGCTTCTGTTTTATTTTGT	202
RPA-MG-F3	ATTTGGTCCTAATCCCCAACAAAGAATTAACCC	301
RPA-MG-R3	ATTAAACCCACCTCCAGCTTTATTTCCCATCG	301
RPA-MS-F1	AAAATTAAATTACTATTAGCAGCTAGTGCAGT	130
RPA-MS-R1	GGTTTTGAGGATTATCAGTATTTGGGTTTCCAG	130
RPA-MS-F2	AATTAAATTACTATTAGCAGCTAGTGCAGT	244
RPA-MS-R2	TTCTGTTTTAGCAGCCTCTACAGGGTCAAC	244
RPA-MS-F3	CCTGAACCAACACCTGGAAACCCAAATACTG	167
RPA-MS-R3	TTAGCTTCTGTTTTAGCAGCCTCTACAGGGTC	167

RPA-MG-F2/R2/μL	RPA-MS-F1/R1/μL
0.0	2.0
0.2	1.8
0.4	1.6
0.6	1.4
0.8	1.2
1.0	1.0
1.2	0.8
1.4	0.6
1.6	0.4
1.8	0.2
2.0	0.0