兔源F型多杀性巴氏杆菌双重PCR检测方法的建立

王锦祥; 孙世坤; 陈岩峰; 陈冬金; 桑雷; 谢喜平

doi:10.19303/j.issn.1008-0384.2021.05.001

兔源F型多杀性巴氏杆菌双重PCR检测方法的建立

doi: 10.19303/j.issn.1008-0384.2021.05.001

福建省农业科学院畜牧兽医研究所，福建　福州　350013

基金项目: 国家现代农业产业技术体系建设专项（CARS-43-G-5）；福建省自然科学基金项目（2002J01346）；福建省科技计划公益类专项（2020R10260015）

详细信息

作者简介:
王锦祥（1983−），男，博士，助理研究员，研究方向：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，研究方向：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
计量
- 文章访问数: 680
- HTML全文浏览量: 178
- PDF下载量: 28
- 被引次数: 0
出版历程
- 收稿日期: 2021-01-14
- 修回日期: 2021-03-23
- 网络出版日期: 2021-04-20
- 刊出日期: 2021-05-31

Duplex PCR Detection on Pasteurella multocida Serogroup F Strain 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F型多杀性巴氏杆菌的双重PCR检测方法，为兔巴氏杆菌病的诊断提供技术支持。方法根据多杀性巴氏杆菌kmt1基因和F型多杀性巴氏杆菌fcbD基因的保守序列分别设计了2对引物进行双重PCR扩增，对双重PCR检测方法的退火温度和混合引物浓度进行优化，并对方法的特异性、敏感性、重复性和准确性进行验证。结果该双重PCR方法最优反应条件为：退火温度60 ℃、混合引物浓度0.8 μmol·L⁻¹。该双重PCR方法能扩增出兔源F型多杀性巴氏杆菌的kmt1基因片段（260 bp）和fcbD基因片段（490 bp）、兔源A和D型多杀性巴氏杆菌的kmt1基因片段（260 bp），对兔源支气管败血波氏杆菌、肺炎克雷伯菌、大肠杆菌、金黄色葡萄球菌和阴性对照均为阴性，无交叉反应，特异性较强。该方法对兔源F型多杀性巴氏杆菌的最低检出限为1×10³拷贝·μL⁻¹，敏感性高。应用该方法对90份病死兔肺脏样品进行批内和批间重复性试验，结果均一致，重复性好。应用该双重PCR方法和已报道的多重PCR方法同时检测87份已知结果的呼吸道病死兔肺脏样品，结果显示双重PCR方法检测结果和已报道的多重PCR方法检测结果与已知结果的符合率分别为97.70%和94.25%，双重PCR方法检测结果与已报道的多重PCR方法检测结果的符合率为93.10%，双重PCR方法的准确性更高。结论针对多杀性巴氏杆菌kmt1基因和F型多杀性巴氏杆菌fcbD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F型多杀性巴氏杆菌的快速检测提供了技术支撑。
- 兔 /
- F型多杀性巴氏杆菌 /
- kmt1基因 /
- fcbD基因 /
- 双重PCR检测方法
Abstract: Objective A duplex PCR assay for rapid detecting Pasteurella multocida serogroup F strain in rabbits was developed. Methods Primer concentration and annealing temperature for the duplex PCR were optimized based upon two sets of specific primers targeting the conserved sequences of kmt1 of P. multocida and fcbD of P. multocida serogroup F strain. Specificity, sensitivity, and accuracy of the methodology were tested for its applicability. Results The optimized conditions for the assay included 0.8 μmol·L⁻¹ on the primer concentration and 60 ℃ for the annealing temperature. The assay detection was specific for P. multocida serogroup A, D, and F strains and free of cross-reactions with Bordetella bronchiseptica, Klebsiella pneumonia, Escherichia coli, Staphylococcus aureus, and negative control. A highly sensitive detection limit up to 1×10³ copies·μL⁻¹ on genomic DNA of P. multocida serogroup F isolate was achieved. In the repeated intra- and inter-assays on 90 lung specimens from dead diseased rabbits, the assay variation coefficients were identical. By using 87 lung samples collected from known infection cases, the newly developed duplex PCR assay and reported multiplex PCR assay yield agreements of 97.70% and 94.25% with the known results, respectively. Moreover, the current duplex PCR determination also yielded an agreement of 93.10% on the measurements with the reported from a multiplex PCR assay. Conclusion The newly established duplex PCR assay based on the specific primers targeting the conserved sequences of kmt1 of P. multocida as well as fcbD of P. multocida serogroup F strain was tested to be highly specific, sensitive, repeatable, and accurate in detecting the pathogenic P. multocida serogroup F strain in rabbits.
- rabbit /
- Pasteurella multocida serogroup F strain /
- kmt1 gene /
- fcbD gene /
- duplex PCR assay

HTML全文

图 1 单重PCR扩增结果

注：M:DNA Marker; 1:kmt1基因; 2:fcbD基因; 3: kmt1基因阴性对照; 4: fcbD基因阴性对照。

Figure 1. Detection by single PCR amplification

Note: M: DNA marker; 1: kmt1 gene; 2: fcbD gene; 3: negative control of kmt1 gene; 4: negative control of fcbD gene.

下载: 全尺寸图片幻灯片

图 2 双重PCR扩增结果

注：M:DNA Marker; 1:kmt1和fcbD基因; 2:阴性对照。

Figure 2. Detection by duplex PCR amplification

Note: M: DNA marker; 1: kmt1 and fcbD genes; 2: negative control.

下载: 全尺寸图片幻灯片

图 3 双重PCR方法反应温度的优化

注：M: DNA Marker; 1: 54 ℃; 2: 54.4 ℃; 3: 55.2 ℃; 4：56.4 ℃; 5：57.8 ℃; 6：59 ℃; 7：59.7 ℃; 8：60 ℃。

Figure 3. Optimization on reaction temperature for duplex PCR assay

Note: M: DNA marker; 1: 54 ℃; 2: 54.4 ℃; 3: 55.2 ℃; 4: 56.4 ℃; 5: 57.8 ℃; 6: 59 ℃; 7: 59.7 ℃; 8: 60 ℃.

下载: 全尺寸图片幻灯片

图 4 双重PCR检测方法引物浓度优化

注：M：DNA Marker; 1～7分别为：0.4、0.5、0.6 、0.7、0.8、0.9、1.0 μmol·L⁻¹。

Figure 4. Optimization on primer concentration for duplex PCR assay

Note: M: DNA marker; 1-7: 0.4, 0.5, 0.6 , 0.7, 0.8, 0.9, 1.0 μmol·L⁻¹.

下载: 全尺寸图片幻灯片

图 5 双重PCR检测方法特异性试验

注：M： DNA Marker；1：F型多杀性巴氏杆菌；2：A型多杀性巴氏杆菌；3：D型多杀性巴氏杆菌；4：支气管败血波氏杆菌；5：肺炎克雷伯菌；6：大肠杆菌；7：金黄色葡萄球菌；8：阴性对照

Figure 5. Specificity of duplex PCR assay

Note: M: DNA marker; 1: serogroup F strain of P. multocida; 2: serogroup A strain of P. multocida; 3: serogroup D strain of P. multocida; 4:B. bronchiseptica; 5: K. pneumonia; 6: E. coli; 7: S. aureus; 8: negative control.

下载: 全尺寸图片幻灯片

图 6 双重PCR检测方法敏感性试验

注：MM：DNA Marker；1～8分别为：1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, 1×10², 1×10¹, 1×10⁰拷贝·μL⁻¹；9：阴性对照。

Figure 6. Sensitivity of duplex PCR assay

Note: M: DNA marker; 1-8: 1×10⁷ , 1×10⁶ , 1×10⁵ , 1×10⁴, 1×10³, 1×10² , 1×10¹, 1×10⁰ copies·μL⁻¹; 9: negative control.

下载: 全尺寸图片幻灯片

参考文献(14)

[1]	王锦祥, 孙世坤, 陈冬金, 等. 一株致兔结膜炎多杀性巴氏杆菌的分离和鉴定 [J]. 中国养兔, 2020(5):9−11. doi: 10.3969/j.issn.1005-6327.2020.05.003 WANG J X, SUN S K, CHEN D J, et al. Isolation and identification of Pasteurella multocida causing conjunctivitis in rabbits [J]. Chinese Journal of Rabbit Farming, 2020(5): 9−11.(in Chinese) doi: 10.3969/j.issn.1005-6327.2020.05.003
[2]	林毅, 李兴玉, 张先慧, 等. 三个品种肉兔人工感染兔巴氏杆菌的抗病性比较研究 [J]. 中国养兔, 2013(6):8−9, 31. doi: 10.3969/j.issn.1005-6327.2013.06.002 LIN Y, LI X Y, ZHANG X H, et al. A comparative study of the difference of the disease resistance to pasterrella multocida among three kinds of rabbits [J]. Chinese Journal of Rabbit Farming, 2013(6): 8−9, 31.(in Chinese) doi: 10.3969/j.issn.1005-6327.2013.06.002
[3]	TOWNSEND K M, BOYCE J D, CHUNG J Y, et al. Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system [J]. Journal of Clinical Microbiology, 2001(39): 924−929.
[4]	GARCÍA-ALVAREZ A, CHAVES F, FERNÁNDEZ A, et al. An ST11 clone of Pasteurella multocida, widely spread among farmed rabbits in the Iberian Peninsula, demonstrates respiratory niche association [J]. Infection Genetics and Evolution, 2015(34): 81−87.
[5]	WANG J X, SANG L, SUN S K, et al. Characterization of Pasteurella multocida isolated from dead rabbits with respiratory disease in Fujian, China [J]. BMC Veterinary Research, 2019, 15(1): 438. doi: 10.1186/s12917-019-2191-3
[6]	RIMLER R B, RHOADES K R. Serogroup F, a new capsule serogroup of Pasteurella multocida [J]. Journal of Clinical Microbiology, 1987, 25(4): 615−618. doi: 10.1128/JCM.25.4.615-618.1987
[7]	RHOADES K R, RIMLER R B. Capsular groups of Pasteurella multocida isolated from avian hosts [J]. Avian Diseases, 1987, 31(4): 895−898. doi: 10.2307/1591048
[8]	JONAS M, MORISHITA T Y, ANGRICK E J., et al Characterization of nine Pasteurella multocida isolates from avian cholera outbreaks in Indonesia [J]. Avian Diseases, 2001, 45(1): 34−42. doi: 10.2307/1593009
[9]	MASSACCI F R, MAGISTRALI C F, CUCCO L, et al. Characterization of Pasteurella multocida involved in rabbit infections [J]. Veterinary Microbiology, 2018, 213: 66−72. doi: 10.1016/j.vetmic.2017.11.023
[10]	张娜, 李书光, 陈金龙, 等. 兔F型多杀性巴氏杆菌的分离鉴定及其致病性 [J]. 中国兽医科学, 2012, 42(7):685−689. ZHANG N, LI S G, CHEN J L, et al. Isolation, identification and pathogenicity of Pasteurella multocida serogroup F from rabbits [J]. Veterinary Science in China, 2012, 42(7): 685−689.(in Chinese)
[11]	JAGLIC Z, JEKLOVA E, LEVA L, et al. Experimental study of pathogenicity of Pasteurella multocida serogroup F in rabbits [J]. Veterinary Microbiology, 2008, 126(1-3): 168−177. doi: 10.1016/j.vetmic.2007.06.008
[12]	王锦祥, 桑雷, 孙世坤, 等. 一株兔源多杀性巴氏杆菌的分离鉴定及荚膜血清分型 [J]. 中国养兔, 2019(1):4−6. WANG J X, SANG L, SUN S K, et al. Identification and capsule serotyping of a Pasteurella multocida isolated from rabbit [J]. Chinese Journal of Rabbit Farming, 2019(1): 4−6.(in Chinese)
[13]	许腾林, 邢桂玲, 刘家森, 等. 多杀性巴氏杆菌TaqMan荧光定量PCR检测方法的建立与应用 [J]. 中国预防兽医学报, 2018, 40(8):706−710. XU T L, XING G L, LIU J S, et al. Development and application of TaqMan real-time PCR assay for the detection of Pasteurella multocida [J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(8): 706−710.(in Chinese)
[14]	唐先春, 吴斌, 刘国平, 等. 应用双重PCR检测产毒素多杀性巴氏杆菌 [J]. 中国兽医学报, 2005, 25(4):374−375. doi: 10.3969/j.issn.1005-4545.2005.04.013 TANG X C, WU B, LIU G P, et al. Detection of toxigenic Pasteurella multocida by Diplex pcr [J]. Chinese Journal of Veterinary Science, 2005, 25(4): 374−375.(in Chinese) doi: 10.3969/j.issn.1005-4545.2005.04.013