兔源强毒力金黄色葡萄球菌双重PCR检测方法的建立

王锦祥; 孙世坤; 陈岩锋; 陈冬金; 桑雷; 谢喜平

doi:10.19303/j.issn.1008-0384.2020.08.007

兔源强毒力金黄色葡萄球菌双重PCR检测方法的建立

doi: 10.19303/j.issn.1008-0384.2020.08.007

福建省农业科学院畜牧兽医研究所，福建　福州　350013

基金项目: 福建省科技计划公益类专项（2019R1026-9）；国家兔产业技术体系建设专项（CARS-43-G-5）

详细信息

作者简介:
王锦祥（1983−），男，助理研究员，主要从事畜禽疫病防控技术研究（E-mail：wjx841227@126.com）

通讯作者:
谢喜平（1966−），男，研究员，主要从事草食动物育种、饲养技术研究与示范推广（E-mail：xxp702@163.com）

中图分类号: S 852
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出版历程
- 收稿日期: 2020-05-21
- 修回日期: 2020-07-30
- 刊出日期: 2020-08-19

A Duplex PCR Assay for Simultaneously Detecting Two Virulent Strains of Staphylococcus aureus in Rabbits

Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou, Fujian　350013, China

摘要

摘要: 目的建立检测兔源强毒力金黄色葡萄球菌（Staphylococcus aureus）的双重PCR检测方法，为兔葡萄球菌病的诊断提供技术支持。方法根据兔源金黄色葡萄球菌的nuc和pvl两种毒力基因的保守序列分别设计了两对特异性引物进行双重PCR扩增，对双重PCR反应体系的混合引物浓度和退火温度进行优化，对双重PCR检测方法的特异性、敏感性和准确性进行验证。结果当双重PCR反应体系混合引物的浓度为0.6 μmol·L⁻¹、退火温度为59.6 ℃时，双重PCR的扩增效果最优。该双重PCR检测方法特异性好，能扩增出兔源强毒力金黄色葡萄球菌的nuc（320 bp）和pvl（585 bp）基因片段以及低毒力菌株的nuc（320 bp）基因片段，对兔源多杀性巴氏杆菌、支气管败血波氏杆菌、肺炎克雷伯菌和大肠杆菌等4种常见兔源细菌性病原菌以及阴性对照均无交叉反应。该方法敏感性高，能分别检出10 pg和100 fg的强毒力和低毒力兔源金黄色葡萄球菌基因组DNA。该方法重复性好，批内和批间重复性试验的变异系数均为0；该方法准确性好，对119份临床样品的检测结果与已报道的检测金黄色葡萄球菌nuc和pvl基因的单重PCR检测方法的符合率为100%。结论针对nuc和pvl两种毒力基因建立的双重PCR检测方法具有良好的特异性、敏感性、重复性和准确性，为兔源强毒力金黄色葡萄球菌的检测提供了有效的技术支持。
- 兔 /
- 金黄色葡萄球菌 /
- 强毒力菌株 /
- 低毒力菌株 /
- 双重PCR检测方法
Abstract: Objective A duplex PCR assay for simultaneous detection on hyper- and low-virulent Staphylococcus aureus strains in rabbits was developed. Methods The assay was based on the specific primers targeting the nuc and pvl genes in a hyper-virulent and a low-virulent strain of S. aureus isolated from rabbits in Fujian. Primer concentration and annealing temperature for the assay were optimized, and detection specificity, sensitivity, and accuracy determined. Results The optimized primer concentration was 0.6 μmol·L⁻¹ and annealing temperature 59.6 ℃ for the assay. Only the hyper- and low-virulent S. aureus isolated from rabbits were detected with no cross-reactions on the commonly found pathogens, such as Pasteurella multocida , Bordetella bronchiseptica, Klebsiella pneumonia , and Escherichia coli , nor on the negative control. The methodology was highly sensitive with a detection limit of 10 pg genomic DNA on the hyper-virulent strain and 100 fg on the low-virulent strains. The coefficients of variation of intra- and inter-assay were 0 indicating high repeatability on both applications. In addition, the test results of the duplex PCR assay were 100% coincident with those obtained by the PCR assays targeting nuc and pvl individually. Conclusion The newly developed duplex PCR assay could simultaneously detect the two virulent S. aureus strains with high specificity, sensibility, repeatability, and accuracy.
- rabbit /
- Staphylococcus aureus /
- hyper-virulent strain /
- low-virulent strain /
- duplex PCR assay

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图 1 单重PCR扩增结果

注：M:DNA Marker；1：强毒力菌株nuc基因；2：强毒力菌株pvl基因；3：低毒力菌株nuc基因；4：低毒力菌株pvl基因；5：阴性对照nuc基因；6：阴性对照pvl基因。

Figure 1. Result of single PCR amplification

Note: M: DNA marker; 1: nuc of hyper-virulent strain; 2: pvl of hyper-virulent strain; 3: nuc of low-virulent strain; 4: pvl of low-virulent strain; 5: nuc of negative control; 6: pvl of negative control.

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图 2 双重PCR扩增结果

注：M：DNA Marker；1：强毒力菌株；2：低毒力菌株；3：阴性对照。

Figure 2. Result of duplex PCR amplification

Note: M: DNA marker; 1: hyper-virulent strain; 2: low-virulent strain; 3: negative control.

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图 3 双重PCR检测方法引物浓度优化

注：A：强毒力菌株；B：低毒力菌株；M：DNA Marker；1~8表示引物浓度为0.2、0.3、0.4 、0.5、0.6、0.7、0.8 μmol·L⁻¹。

Figure 3. Optimization on primer concentration for duplex PCR assay

Note: A: hyper-virulent strain; B: low-virulent strain; M: DNA marker; 1–8: primer concentration 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 μmol·L⁻¹.

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图 4 双重PCR检测方法退火温度优化

注：A：强毒力菌株；B：低毒力菌株；M：DNA Marker；1~8表示退火温度53.0、53.5、54.4、55.8、57.5、58.8、59.6、60.0 ℃。

Figure 4. Optimization on annealing temperature for duplex PCR assay

Note: A: hyper-virulent strain; B: low-virulent strain; M: DNA marker; 1–8: annealing temperature at 53, 53.5, 54.4, 55.8, 57.5, 58.8, 59.6 and 60 ℃.

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图 5 双重PCR检测方法特异性

注：M：DNA Marker；1：强毒力菌株；2：低毒力菌株；3：多杀性巴氏杆菌；4：支气管败血波氏杆菌；5：肺炎克雷伯菌；6：大肠杆菌；7：阴性对照。

Figure 5. Specificity test of duplex PCR assay

Note: M: DNA marker; 1: hypre-virulent strain; 2: low-virulent strain; 3: Pasteurella multocida; 4: Bordetella bronchiseptica; 5: Klebsiella pneumonia; 6: Escherichia coli; 7: negative control.

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图 6 双重PCR检测方法敏感性

注：A：强毒力菌株；B：低毒力菌株；M：DNA Marker；1~9：100 ng、10 ng、1 ng、100 pg、10 pg、1 pg、100 fg、10 fg、1 fg。

Figure 6. Sensitivity test of duplex PCR assay

Note: A: hyper-virulent strain; B: low-virulent strain; M: DNA marker; 1: 100 ng; 2: 10 ng; 3: 1 ng; 4: 100 pg; 5: 10 pg; 6: 1 pg; 7: 100 fg; 8: 10 fg; 9: 1 fg.

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表 1 双重PCR检测方法引物

Table 1. Primers for duplex PCR assay

靶基因 Target genes	引物名称 Primers	引物序列（5′-3′） Primer sequences（5′-3′）	产物大小 Product size/bp
nuc	nuc-F	acttcaactaaaaaattacataaagaacct	320
	nuc-R	aagccttgacgaactaaagct
pvl	pvl-F	gcaacatcagattccgataagtta	585
	pvl-R	ggataacactggcattttgtga

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表 2 临床样品的检测

Table 2. Detection of clinical samples

样品 Samples	单重PCR检测结果 Results of single PCR/份	双重PCR检测结果 Results of duplex PCR/份	符合率 Coincidence/%
强毒力菌株 Hyper-virulent strain	30	30	100
低毒力菌株 Low-virulent strain	45	45	100
阴性 Negative	44	44	100
总计 Control	119	119	100

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参考文献(11)

[1]	赵蕾, 李坤林, 李书光, 等. 长毛兔金黄色葡萄球菌的分离鉴定 [J]. 中国畜牧兽医, 2011, 38(9):184−186. ZHAO L, LI K L, LI S G, et al. Isolation and identification of a staphylococcus aureus from rabbit [J]. China Animal Husbandry & Veterinary Medicine, 2011, 38(9): 184−186.(in Chinese)
[2]	王锦祥, 桑雷, 孙世坤, 等. 一株兔源金黄色葡萄球菌的分离和鉴定 [J]. 中国养兔, 2019(3):4−6. doi: 10.3969/j.issn.1005-6327.2019.03.001 WANG J X, SANG L, SUN S K, et al. Isolation and identification of a Staphylococcus aureus from rabbit [J]. Chinese Journal of Rabbit Farming, 2019(3): 4−6.(in Chinese) doi: 10.3969/j.issn.1005-6327.2019.03.001
[3]	王锦祥, 孙世坤, 陈岩峰, 等. 一株致兔皮下脓肿金黄色葡萄球菌的分离鉴定 [J]. 中国养兔, 2019(5):18−20. doi: 10.3969/j.issn.1005-6327.2019.05.004 WANG J X, SUN S K, CHEN Y F, et al. Isolation and identification of a Staphylococcus aureus causing subcutaneous abscess in rabbit [J]. Chinese Journal of Rabbit Farming, 2019(5): 18−20.(in Chinese) doi: 10.3969/j.issn.1005-6327.2019.05.004
[4]	WANG J X, SANG L, CHEN Y F, et al. Characterisation of Staphylococcus aureus strain causing severe respiratory disease in rabbits [J]. World Rabbit Science, 2019, 27(1): 41−48. doi: 10.4995/wrs.2019.10454
[5]	张国权, 孙乐天, 王国艳, 等. 獭兔源金黄色葡萄球菌16S rDNA鉴定与耐药性分析 [J]. 山西农业科学, 2018, 46(11):1926−1928, 1942. doi: 10.3969/j.issn.1002-2481.2018.11.36 ZHANG G Q, SUN L T, WANG G Y, et al. Identification of 16S rDNA of Staphylococcus aureus from rex rabbits and analysis of drug resistance [J]. Journal of Shanxi Agricultural Sciences, 2018, 46(11): 1926−1928, 1942.(in Chinese) doi: 10.3969/j.issn.1002-2481.2018.11.36
[6]	WANG J, SANG L, SUN S, et al. Characterisation of Staphylococcus aureus isolated from rabbits in Fujian, China [J]. Epidemiology and Infection, 2019, 147: e256. doi: 10.1017/s0950268819001468
[7]	邓钊宾, 余滔, 耿毅, 等. 兔源金黄色葡萄球菌的耐药性及耐药基因分析 [J]. 中国预防兽医学报, 2016, 38(1):45−48. doi: 10.3969/j.issn.1008-0589.2016.01.11 DENG Z B, YU T, GENG Y, et al. Antibiotic resistance phenotype and genes detection of Staphylococcus aureus isolated from rabbit [J]. Chinese Journal of Preventive Veterinary Medicine, 2016, 38(1): 45−48.(in Chinese) doi: 10.3969/j.issn.1008-0589.2016.01.11
[8]	BRAKSTAD O G, AASBAKK K, MAELAND J A. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene [J]. Journal of Clinical Microbiology, 1992, 30(7): 1654−1660. doi: 10.1128/JCM.30.7.1654-1660.1992
[9]	LABANDEIRA-REY M, COUZON F, BOISSET S, et al. Staphylococcus aureus panton-valentine leukocidin causes necrotizing pneumonia [J]. Science, 2007, 315(5815): 1130−1133. doi: 10.1126/science.1137165
[10]	向正刚, 耿毅, 张雨薇, 等. 四川兔源金黄色葡萄球菌毒力检测及分子分型 [J]. 华南农业大学学报, 2017, 38(4):62−68. doi: 10.7671/j.issn.1001-411X.2017.04.011 XIANG Z G, GENG Y, ZHANG Y W, et al. Virulence detection and molecular typing of Staphylococcus aureus isolated from rabbit in Sichuan area [J]. Journal of South China Agricultural University, 2017, 38(4): 62−68.(in Chinese) doi: 10.7671/j.issn.1001-411X.2017.04.011
[11]	RAO Q, SHANG W L, HU X M, et al. Staphylococcus aureus ST121: a globally disseminated hypervirulent clone [J]. Journal of Medical Microbiology, 2015, 64(12): 1462−1473. doi: 10.1099/jmm.0.000185