丝状支原体簇和多杀性巴氏杆菌双重PCR方法的建立及应用

Abstract

Abstract: The aim of this study was to establish a duplex PCR method for simultaneous detection of Mycoplasma mycoides cluster and Pasteurella multocida, so as to provide a rapid, accurate and convenient detection tool for these pathogens. Two pairs of primers which were specific to Mycoplasma mycoides cluster and Pasteurella multocida，respectively, were used to develop the method. Following optimization of PCR components and reaction profile, specificity and sensitivity of the assay were evaluated. A total of 52 clinical samples were tested using the duplex PCR. The results showed that the assay could specifically amplify Mycoplasma mycoides cluster and Pasteurella multocida, while no-target pathogens were not detected. The detection limits of the assay were determined to be 24.8 and 28.9 pg for Mycoplasma mycoides cluster and Pasteurella multocida, respectively. The duplex PCR could detect Mycoplasma mycoides cluster and Pasteurella multocida from clinical samples. These results suggested that the duplex PCR assay was specific and sensitive, and would be useful for clinical detection, identification and epidemiological investigation of Mycoplasma mycoides cluster and Pasteurella multocida.

Key words: Mycoplasma mycoides cluster; Pasteurella multocida; duplex PCR

WANG Cheng-long, WU Yu-xi, FENG Xu-fei, YANG Fa-long. Development and Application of a Duplex PCR Assay for the Detection of Mycoplasma mycoides Cluster and Pasteurella multocida[J]. .

References

1 Bascunana C R, Mattsson J G, Bolske G, et al. Characterization of the 16S rRNA genes from Mycoplasma sp. strain F38 and development of an identification system based on PCR［J］. J Bacteriol, 1994, 176(9): 2577～2586.
2 Besser T E, Highland M A, Baker K, et al. Causes of pneumonia epizootics among bighorn sheep, Western United States, 2008—2010［J］. Emerg Infect Dis, 2012, 18(3): 406～414.
3 Dziva F, Muhairwa A P, Bisgaard M, et al. Diagnostic and typing options for investigating diseases associated with Pasteurella multocida［J］. Vet Microbiol, 2008, 128(1): 1～22.
4 Fablet C, Marois C, Dorenlor V, et al. Bacterial pathogens associated with lung lesions in slaughter pigs from 125 herds［J］. Res Vet Sci, 2012, 93(2): 627～630.
5 Gonalves R, Mariano I, Núez A, et al. Atypical non-progressive pneumonia in goats［J］. The Veterinary Journal, 2010, 183(2): 219～221.
6 Kalorey D R, Yuvaraj S, Vanjari S S, et al. PCR analysis of Pasteurella multocida isolates from an outbreak of pasteurellosis in Indian pigs［J］. Comp Immunol Microbiol Infect Dis, 2008, 31(6): 459～465.
7 McAuliffe L, Hatchell F M, Ayling R D, et al. Detection of Mycoplasma ovipneumoniae in Pasteurella-vaccinated sheep flocks with respiratory disease in England［J］. Vet Rec, 2003, 153(22): 687～688.
8 Rhoades K R, Rimler R B. Capsular groups of Pasteurella multocida isolated from avian hosts［J］. Avian Dis, 1987, 31: 895～898.
9 Rodriguez J L, Ermel R W, Kenny T P, et al. Polymerase chain reaction and restriction endonuclease digestion for selected members of the “Mycoplasma mycoides cluster” and Mycoplasma putrefaciens［J］. J Vet Diagn Invest, 1997, 9(2): 186～190.
10 Shahram M, Nicholas R A, Wood A P, et al. Further evidence to justify reassignment of Mycoplasma mycoides subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri［J］. Syst Appl Microbiol, 2010, 33(1): 20～24.
11 Thiaucourt F, Bolske G. Contagious caprine pleuropneumonia and other pulmonary mycoplasmoses of sheep and goats［J］. Rev Sci Tech, 1996, 15(4): 1397～1414.
12 Townsend K M, Frost A J, Lee C W, et al. Development of PCR assays for species- and type-specific identification of Pasteurella multocida isolates［J］. J Clin Microbiol, 1998, 36(4): 1096～1100.
13 Woubit S, Manso-Silvan L, Lorenzon S, et al. A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: Application to the diagnosis of contagious agalactia［J］. Mol Cell Probes, 2007, 21(5): 391～399.

[1]	ZHANG Yundan, MA Guangqiang, YUE Jun, ZHOU Bijun, WEN Ming, CHENG Zhentao. Establishment and Application of Duplex PCR for Rapid Identification of Staphylococcus and Streptococcus of Livestock and Poultry in Guizhou Province [J]. , 2019, 46(3): 873-880.
[2]	HE Ying, XIE Zhi-xun, LUO Si-si, LI Meng, XIE Li-ji, DENG Xian-wen, XIE Zhi-qin, FENG Bin, HUANG Jiao-ling, ZHANG Yan-fang. Development of a Duplex PCR Assay for Detection of Avian Influenza Virus and Chicken Parvovirus [J]. , 2017, 44(7): 2126-2131.
[3]	LIAN Si-nan, LIANG Lin, BAI Ai-quan, CUI Shang-jin. Establishment and Primary Application of Duplex PCR Assay to Detect Procine Pseudorabies Virus [J]. , 2016, 43(5): 1169-1175.
[4]	ZHENG Jia-qi, HUANG Hai-bi, WANG Xiao-hui, LI Zhen-ya, XING Meng-en, HAO Yong-qing. Development and Application of a Duplex PCR Assay for Detecting Mycoplasma ovipneumiae and Mycoplasma arginini [J]. , 2015, 42(9): 2240-2245.
[5]	ZHAO Xue-li, YAN Ruo-qian, WU Zhi-ming, CAO Wei-wei, WANG Shu-juan, XIE Cai-hua, MA Zhen-yuan, ZHOU Bing-qiang, WANG Dong-fang. Establishment and Application of a Duplex PCR Assay for Detection of Pseudorabies Virus (PRV) gE-deleted Vaccine and PRV [J]. , 2015, 42(4): 865-870.
[6]	PENG Wu-li, LUO Zhan-rong, SHI Qian, YUN Li-juan, YU Xue-hui, JI Xin-cheng. Establishment and Application of a Duplex PCR Method for Detecting Brucella and Chlamydia psittaci [J]. , 2014, 41(4): 7-11.
[7]	SUN Yuan-yuan, ZHAO Peng, LIU Jun, WANG Yun, QIAN Ke. Development and Application of Duplex PCR for Canine Distemper Virus and Canine Coronavirus Detection [J]. , 2014, 41(3): 99-103.
[8]	ZHAO Hong, XIE Zhi-xun, XIE Li-ji, LIU Jia-bo, DENG Xian-wen, XIE Zhi-qin, PANG Yao-shan, FAN Qing, LUO Si-si. Establishment of Duplex PCR Assay for Detection of H9 Subtype Avian Influenza Virus and Duck Tembusu Virus [J]. , 2014, 41(10): 43-47.
[9]	ZHANG Yan-fang, XIE Zhi-xun, XIE Li-ji, LIU Jia-bo, FAN Qing, PANG Yao-shan, DENG Xian-wen, XIE Zhi-qin. Establishment of a Duplex PCR Assay for Detection of Duck Newcastle Disease Virus and Muscovy Duck Parvovirus [J]. , 2013, 40(11): 63-66.
[10]	CHEN An-li, XIE Zhi-xun, XIE Li-ji, XIE Zhi-qin, PANG Yao-shan, DENG Xian-wen, LIU Jia-bo, PENG Yi, FAN Qin, LUO Si-si. Development and Application of a Duplex PCR Assay for Detection of Newcastle Diseases Virus and Duck Plague Virus [J]. , 2013, 40(11): 192-195.
[11]	ZHANG Zhen-guo;XU Li-pu;WANG Shu;WANG Jing-bo;WANG Xiao-liang;CAO Huan. Detection of Koi Herpesvirus by Duplex PCR Assay [J]. , 2011, 38(9): 83-86.
[12]	XIANG Zhi-long;CHENG Zhen-tao;ZHUO Jian-hua;ZHOU Bi-jun;OU De-yuan;XIAN Si-mei;LIU Fang;RAN Guang-xin. Establishment and Rudiment Application of Duplex PCR for Goatpox Virus and Orf Virus [J]. , 2011, 38(1): 88-91.

Development and Application of a Duplex PCR Assay for the Detection of Mycoplasma mycoides Cluster and Pasteurella multocida

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 12

Recommended Articles

Metrics

Comments