Duplex PCR Detection on <i>Pasteurella multocida</i> Serogroup F Strain in Rabbits

WANG Jinxiang; SUN Shikun; CHEN Yanfeng; CHEN Dongjin; SANG Lei; XIE Xiping

doi:10.19303/j.issn.1008-0384.2021.05.001

Volume 36 Issue 5

May 2021

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Article Navigation > Fujian Journal of Agricultural Sciences > 2021 > 36(5): 501-505

WANG J X, SUN S K, CHEN Y F, et al. Duplex PCR Detection on Pasteurella multocida Serogroup F Strain in Rabbits [J]. Fujian Journal of Agricultural Sciences，2021，36（5）：501−505 doi: 10.19303/j.issn.1008-0384.2021.05.001

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WANG J X, SUN S K, CHEN Y F, et al. Duplex PCR Detection on Pasteurella multocida Serogroup F Strain in Rabbits [J]. Fujian Journal of Agricultural Sciences，2021，36（5）：501−505 doi: 10.19303/j.issn.1008-0384.2021.05.001

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Duplex PCR Detection on Pasteurella multocida Serogroup F Strain in Rabbits

doi: 10.19303/j.issn.1008-0384.2021.05.001

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

Received Date: 2021-01-14
Rev Recd Date: 2021-03-23

Available Online: 2021-04-20

Publish Date: 2021-05-31

Abstract

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

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References(14)

References

[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