绵羊肺炎支原体和山羊伪结核棒状杆菌双重PCR检测方法的建立及应用

黄丽丽; 林裕胜; 肖义军; 胡奇林

doi:10.19303/j.issn.1008-0384.2020.01.007

绵羊肺炎支原体和山羊伪结核棒状杆菌双重PCR检测方法的建立及应用

doi: 10.19303/j.issn.1008-0384.2020.01.007

黄丽丽^{1, 2,},
林裕胜²,
肖义军¹,
胡奇林^2, ,

1.
福建师范大学生命科学学院，福建　福州　350108
2.
福建省农业科学院畜牧兽医研究所，福建　福州　350013

基金项目: 国家重点研发计划项目（2016YFD0500906）；福建省农业科学院科技创新团队建设项目（STIT2017-1-5）；福建省科技计划公益类专项（2018R1023-1）

详细信息

作者简介:
黄丽丽（1991−），女，硕士研究生，主要从事动物传染病研究（E-mail：huanglilixj2012@163.com）

通讯作者:
胡奇林（1963−），男，研究员，主要从事动物传染病学和免疫学研究（E-mail：hql562713@163.com）

中图分类号: S 852.62
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出版历程
- 收稿日期: 2019-10-15
- 修回日期: 2019-12-25
- 刊出日期: 2020-01-01

A New Multiplex PCR Assay for Simultaneously Detecting Mycoplasma ovipneumoniae and Corynebacterium pseudotuberculosis in Diseased Goats and Sheep

HUANG Li-li^{1, 2
,},
LIN Yu-sheng²,
XIAO Yi-jun¹,
HU Qi-lin^{2
, ,}

1.
College of Life Sciences, Fujian Normal University, Fuzhou, Fujian　350108, China
2.
Institute of Animal Husbandry and Veterinary, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China

摘要

摘要: 目的为实现临床上对绵羊肺炎支原体（Mycoplasma ovipneumoniae, Mo）和山羊伪结核棒状杆菌（Corynebacterium pseudotuberculosis, CP）两种病原混合感染的同时检测，建立Mo和CP的双重PCR检测方法。方法应用基于Mo的P80基因和CP的PLD基因设计两对特异性引物，对反应体系和条件进行优化，验证方法的特异性、灵敏性和重复性；应用该方法和单一PCR方法对临床收集的70份样品进行检测，比较检测结果。结果建立的Mo和CP的双重PCR检测方法能够同时扩增出Mo 700 bp和CP 290 bp左右的片段，而对其他羊病常见病原的DNA扩增均为阴性。Mo和CP的检测下限分别为1 530 pg·μL⁻¹和3 500 pg·μL⁻¹。该方法具有良好的重复性。临床样品检测结果显示：70份临床样品中Mo阳性率为25.71%，CP阳性率为18.57%，同时感染Mo和CP阳性率为7.14%，与单一PCR检测结果符合率为100%。结论本研究建立的双重PCR检测方法具有特异性强、灵敏性较高、重复性好等特点，为临床上Mo和CP混合感染的快速检测以及流行病学调查提供了实用方法。
- 绵羊肺炎支原体 /
- 伪结核棒状杆菌 /
- 双重PCR
Abstract: Objective To establish a multiplex PCR method for simultaneous clinic detection of Mycoplasma ovipneumoniae (Mo), one of the main pathogens causing Mycoplasma pneumonia of goats and sheep(MPGS), and Corynebacterium pseudotuberculosis (CP), the pathogen of goat pseudotuberculosis, in the livestock animals infected by both pathogens at a same time. Method Two pairs of specific primers were designed based on the P80 gene of Mo and the PLD gene of CP for the PCR. After optimizing the detection system and reaction conditions, the specificity, sensitivity, and repeatability of the new method were verified for methodology validation. The results obtained using two separate PCR and the multiplex PCR on 70 clinical specimens were comparatively analyzed. Result The newly developed multiplex PCR method amplified a 700 bp fragment from Mo and a 290 bp fragment from CP, with none from other common pathogens of goats and sheep. The method had a detection limit of 1 530 pg·μL⁻¹ on Mo and 3 500 pg·μL⁻¹ on CP with a high repeatability. A test on 70 clinical specimens using the new method yielded a 25.71% positive rate on Mo, 18.57% on CP, and 7.14% on Mo/CP infections. Its coincidence rate with the two single PCR results was 100%. Conclusion The newly established multiplex PCR detection methodology exhibited high specificity, sensitivity, and reproducibility. It appeared to be applicable for rapid and simultaneous clinic detection on Mo and CP as well as epidemiological studies on the disease infected by the pathogens.
- Mycoplasma ovipneumoniae /
- Corynebacterium pseudotuberculosis /
- multiplex PCR

HTML全文

图 1 Mo和CP单一和双重PCR扩增

注：M: 2 000 DNA Ladder；1: Mo和CP；2：Mo；3: CP；4: 阴性对照。

Figure 1. Single and multiplex PCR amplifications of Mo and CP

Note：M: 2 000 DNA Ladder; 1: Mo and CP; 2: Mo; 3: CP; 4: negative control.

下载: 全尺寸图片幻灯片

图 2 双重PCR特异性试验

注：M：2 000 DNA Ladder；1：Mo+CP；2–11分别为CP、Mo、Mmc、Mcc、Mccp、AL、Mb、SE、Ec、SA；12: 空白对照。

Figure 2. Specificity of multiplex PCR

Note：M: 2 000 DNA Ladder; 1: Mo and CP; 2–11: CP, Mo, Mmc, Mcc, Mccp, AL, Mb, SE, Ec, and SA, respectively; 12: blank control.

下载: 全尺寸图片幻灯片

图 3 双重PCR灵敏性试验

注：M: 2 000 DNA Ladder；1: Mo＋CP阳性对照；2–9: 分别为10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸倍稀释的含Mo和CP的阳性样本；10: 阴性对照。

Figure 3. Sensitivity of multiplex PCR

Note：M: 2 000 DNA Ladder; 1: Mo and CP positive control; 2–9: positive specimens containing Mo and CP diluted 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ times, respectively; 10: negative control.

下载: 全尺寸图片幻灯片

图 4 单一PCR灵敏性试验

注：A中，M: 2 000 DNA Ladder；1: Mo阳性对照；2–9: 分别为10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸倍稀释的含Mo的阳性样本；10: 阴性对照；B中，M: 2 000 DNA Ladder；1: CP 阳性对照；2–9分别为10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸倍稀释的含CP 的阳性样本；10: 阴性对照。

Figure 4. Sensitivity of single PCR

Note：A：M: 2 000 DNA Ladder; 1: Mo positive control; 2–9: positive specimens containing Mo diluted 10¹，10²，10³，10⁴，10⁵，10⁶，10⁷，10⁸ times, respectively; 10: negative control; B: M: 2 000 DNA Ladder; 1: CP positive control; 2–9: positive specimens containing CP diluted 10¹，10²，10³，10⁴，10⁵，10⁶，10⁷，10⁸ times, respectively; 10: negative control.

下载: 全尺寸图片幻灯片

图 5 双重PCR扩增重复性试验

注：M: 2 000 DNA Ladder；1: 阳性对照；2–5: Mo；6–10: CP；11–16分别为Mccp、Mmc、AL、Ec、SA和SE；17: 空白对照。

Figure 5. Repeatability of multiplex PCR

Note：M: 2 000 DNA Ladder; 1: positive control; 2–5: Mo; 6–10: CP; 11–16: Mccp, Mmc, AL, Ec, SA, and SE, respectively; 17: blank control.

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

靶基因 Target gene	引物名称 Primer name	引物序列（5′→ 3′） Primer sequence（5′→ 3′）	扩增片段大小 Amplified fragment size/bp
P80基因	F1	GCCTTGGGGTTGGAATTCCTTTGTCTTATTC	705
P80基因	R1	CATTTGATGCTGAGGTCGGATTTGGACTAAC	705
PLD基因	F2	GTGAGAAGAACCCCGGTATAAG	291
PLD基因	R2	TACCGCACTTATTCTGACACTG	291

下载: 导出CSV

表 2 临床样品检测结果

Table 2. Detection of pathogens on clinical specimens

样品名称 Sample name	Mo阳性率 Mo positive rate/%	CP阳性率 CP positive rate/%	Mo + CP阳性率 Mo + CP positive rate/%
鼻拭子 Nasal cotton swab	18.60	11.63	4.65
肺组织 Lung tissue	37.04	29.63	11.11
所有样品 All samples	25.71	18.57	7.14

下载: 导出CSV

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