鸡毒支原体和滑液囊支原体双重LFD-RPA快速检测方法的建立

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

摘要/Abstract

摘要：

【目的】 鸡毒支原体（Mycoplasma gallisepticum, MG）和滑液囊支原体（Mycoplasma synoviae, MS）是对家禽危害最大的两种支原体，MG、MS的早期诊断是预防和控制禽支原体的关键环节。通过将重组酶聚合酶扩增技术（recombinase polymerase amplification, RPA）和测流层析试纸条（lateral flow dipstick, LFD）相结合，建立一种操作便捷、反应快速、结果可视化、可同时检测MG、MS两种病原的快速检测方法。【方法】 基于MG的mgc2基因、MS的vlhA基因设计特异性引物和探针，优化反应时间、反应温度及引物配比建立双重LFD-RPA快速检测方法，评价其灵敏度、特异性、稳定性，同时对120份临床样品进行检测。【结果】 双重LFD-RPA检测方法在37℃下反应5-10 min即可完成扩增，其RPA-MG-F2/R2、RPA-MS-F1/R1最佳引物配比为0.6∶1.4。MG、MS的最低检出限分别为3.75 copies/μL、3.46 copies/μL。用该方法与OIE推荐的PCR方法对120份样品进行检测，二者符合率为93.3%。【结论】 MG、MS双重LFD-RPA检测方法在恒温条件下即可完成扩增，其操作简单、反应快速、灵敏度高、特异性强、稳定性好，无需使用任何仪器即可观察到结果，适用于基层MG、MS单独感染或混合感染的现场快速检测。

关键词: 鸡毒支原体, 滑液囊支原体, 重组酶聚合酶扩增技术, 测流层析试纸条

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

田兴苗, 王健霖, 郭磊, 司朵朵, 龚振兴, 李继东. 鸡毒支原体和滑液囊支原体双重LFD-RPA快速检测方法的建立[J]. 生物技术通报, 2024, 40(7): 117-124.

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.

图/表 12

表1 PCR引物

Table 1 PCR primers

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

表2 基础RPA引物

Table 2 Basic RPA primers

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

表3 双重基础RPA引物配比优化

Table 3 Optimization of primer ratios for dual basic RPA

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

表4 LFD-RPA引物及探针

Table 4 LFD-RPA primers and probes

引物/探针名称 Primer/Probe name	引物序列 Primer sequence（5'-3'）
MG-LFD-RPA-F	CCTCCGAACCCTGATTTTATCCAGTAGTG
MG-LFD-RPA-R	TAMRA-GTTGGCTCAATCGCTTCTGTTTTATTTTGT
MG-LFD-RPA-Probe	Digoxin-AGAATGATGATCCAAGAACGTGAAGAACA CCA/idSp/AAGATGGTTGAATCC-C3-spacer
MS-LFD-RPA-F	AAAATTAAATTACTATTAGCAGCTAGTGCAGT
MS-LFD-RPA-R	Biotin-GGTTTTGAGGATTATCAGTATTTGGGTTTCCAG
MS-LFD-RPA-Probe	FAM-TGCTCCTGCTGTTATAGCAATTTCATGTGGTGA /idSp/CAAACTCCAGCACCT-C3-spacer

表5 LFD-RPA反应体系

Table 5 LFD-RPA reaction system（50 μL）

试剂Reagent	体积Volume/μL
MG-LFD-RPA-F	0.6
MG-LFD-RPA-R	0.6
MG-LFD-RPA-Probe	0.3
MS-LFD-RPA-F	1.4
MS-LFD-RPA-R	1.4
MS-LFD-RPA-Probe	0.3
A buffer	29.4
Template	3（MG）+3（MS）
ddH₂O	7.5
B buffer	2.5

图1 试纸条结果判读

Fig. 1 Interpretation of test strip results

表6 OIE PCR引物

Table 6 OIE PCR primers

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	片段大小 Product length/bp
MG 16S rRNA F	GAGCTAATCTGTAAAGTTGGTC	183
MG 16S rRNA R	GCTTCCTTGCGGTTAGCAAC	183
MS 16S rRNA F	GAGAAGCAAAATAGTGATATCA	211
MS 16S rRNA R	CAGTCGTCTCCGAAGTTAACAA	211

图2 PCR扩增结果 M：DL1000 DNA marker；1：MG阴性对照；2：MG PCR产物；3：MS阴性对照；4：MS PCR产物

Fig. 2 PCR amplified results M: DL1000 DNA marker; 1: MG negative control; 2: MG PCR product; 3: MS negative control; 4: MS PCR product

图3 MG、MS基础RPA引物筛选 F1/R1、F2/R2、F3/R3：分别表示第1、2、3对引物；M：DL500 DNA marker；1：5 min；2：10 min；3：15 min；4：20 min；5：25 min；6：30 min

Fig. 3 Primer selection of MG and MS basic RPA F1/R1, F2/R2, F3/R3: The first, second, and third primer pair, respectively;M: DL500 DNA marker; 1: 5 min; 2: 10 min; 3: 15 min; 4: 20 min; 5: 25 min; 6: 30 min

图4 MG、MS双重基础RPA最佳引物配比优化 M：DL500 DNA marker；1-11：RPA-MG-F2/R2、RPA-MS-F1/R1配比分别为0∶2、0.2∶1.8、0.4∶1.6、0.6∶1.4、0.8∶1.2、1∶1、1.2∶0.8、1.4∶0.6、1.6∶0.4、1.8∶0.2、2∶0

Fig. 4 Optimization of optimal primer ratios for MG and MS dual basic RPA M: DL500 DNA marker. 1-11: The ratios of RPA-MG-F2/R2 and RPA-MS-F1/R1 are 0∶2, 0.2∶1.8, 0.4∶1.6, 0.6∶1.4, 0.8∶1.2, 1∶1, 1.2∶0.8, 1.4∶0.6, 1.6∶0.4, 1.8∶0.2, 2∶0, respectively

图5 MG、MS双重LFD-RPA方法的建立 A：反应时间优化；B：反应温度优化；C：灵敏度试验；D：特异性试验；E：重复性试验（a：第1次重复；b：第2次重复；c：第3次重复）；N：阴性对照

Fig. 5 Establishment of dual LFD-RPA method with MG and MS A: Reaction time optimization. B: Reaction temperature optimization. C: Sensitivity test. D: Specificity test. E: Repeatability test（a: The first repeat; b: the second repetition; c: the third repetition）. N: Negative control

表7 MG、MS双重LFD-RPA与OIE PCR检测方法结果比较分析

Table 7 Comparative analysis of MG and MS dual LFD-RPA and OIE PCR detection methods

样品名称 Sample name	LFD-RPA检测结果 LFD-RPA test result	OIE PCR检测结果 OIE PCR test result
MG阳性	37	33
MG阴性	83	87
MS阳性	52	50
MS阴性	68	70

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