猪链球菌通用型和2型双重荧光定量PCR快速检测技术的建立和应用

doi:10.11843/j.issn.0366-6964.2018.02.016

Abstract

Abstract:

In order to detect S. suis and S. suis serotype 2 rapidly, two sets of specific primers and TaqMan probes were designed according to the gdh and cps2J gene of S. suis serotype 2 to establish a duplex real-time PCR assay for detection of S. suis and S. suis serotype 2. Then the duplex real-time PCR was used to detect clinical samples and compare with conventional PCR. Results showed that S. suis and S. suis serotype 2 could be identified in 1.5 hours by the duplex real-time PCR assay and without any cross reaction with other bacteria. Besides, the detection limit was 5 copies/reaction. The correlation coefficient of standard curves was over 0.999, and intra-and inter-assay CV was less than 1.25%. Positive rate for sixty-seven clinical samples was 79.1% and 35.8%, respectively. Coincidence rates of duplex real-time PCR with conventional PCR for S. suis and S. suis serotype 2 were 92.5% and 89.6%, kappa values were 0.800 and 0.757, respectively. A sensitive, specific, reproducible assay is established successfully to detect S. suis and S. suis serotype 2 simultaneously and rapidly, and this assay is very suitable for detection of mass clinical samples.

CLC Number:

S852.611

WU Jing-bo, NAN Wen-jin, HUANG Jian-qiang, HU Hong-hui, PENG Guo-liang, PENG Ling, DONG Xiao-ying. Development of A Rapid Duplex Real-Time PCR Assay for Streptococcus suis and S. suis Serotype 2[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 368-377.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2018.02.016

https://www.xmsyxb.com/EN/Y2018/V49/I2/368

References

[1] GOYETTE-DESJARDINS G, AUGER J P, XU J G, et al. Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing[J]. Emerg Microbes Infect, 2014, 3(6):e45.
[2] GOTTSCHALK M, XU J G, LECOURS M P, et al. Streptococcus suis infections in humans:what is the prognosis for Western countries? (Part Ⅱ)[J]. Clin Microbiol Newslett, 2010, 32(13):97-102.
[3] GOTTSCHALK M, SEGURA M, XU J G. Streptococcus suis infections in humans:the Chinese experience and the situation in North America[J]. Anim Health Res Rev, 2007, 8(1):29-45.
[4] WERTHEIM H F L, NGHIA H D T, TAYLOR W, et al. Streptococcus suis:an emerging human pathogen[J]. Clin Infect Dis, 2009, 48(5):617-625.
[5] ARENDS J P, HARTWIG N, RUDOLPHY M, et al. Carrier rate of Streptococcus suis capsular type 2 in palatine tonsils of slaughtered pigs[J]. J Clin Microbiol, 1984, 20(5):945-947.
[6] MWANIKI C G, ROBERTSON I D, HAMPSON D J. The prevalence of Streptococcus suis type 2 in Western Australian piggeries[J]. Aust Vet J, 1994, 71(11):385-386.
[7] WISSELINK H J, SMITH H E, STOCKHOFE-ZURWIEDEN N, et al. Distribution of capsular types and production of muramidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European countries[J]. Vet Microbiol, 2000, 74(3):237-248.
[8] VECHT U, STOCKHOFE-ZURWIEDEN N, TETENBURG B J, et al. Virulence of Streptococcus suis type 2 for mice and pigs appeared host-specific[J]. Vet Microbiol, 1997, 58(1):53-60.
[9] HIGGINS R, GOTTSCHALK M. An update on Streptococcus suis identification[J]. J Vet Diagn Invest, 1990, 2(3):249-252.
[10] NGA T V T, NGHIA H D T, TU L T P, et al. Real-time PCR for detection of Streptococcus suis serotype 2 in cerebrospinal fluid of human patients with meningitis[J]. Diagn Microbiol Infect Dis, 2011, 70(4):461-467.
[11] MA E, CHUNG P H, SO T, et al. Streptococcus suis infection in Hong Kong:an emerging infectious disease?[J]. Epidemiol Infect, 2008, 136(12):1691-1697.
[12] MESSIER S, LACOUTURE S, GOTTSCHALK M. Distribution of Streptococcus suis capsular types from 2001 to 2007[J]. Can Vet J, 2008, 49(5):461-462.
[13] OKURA M, LACHANCE C, OSAKI M, et al. Development of a two-step multiplex PCR assay for typing of capsular polysaccharide synthesis gene clusters of Streptococcus suis[J]. J Clin Microbiol, 2014, 52(5):1714-1719.
[14] GOTTSCHALK M, LACOUTURE S, BONIFAIT L, et al. Characterization of Streptococcus suis isolates recovered between 2008 and 2011 from diseased pigs in Quebéc, Canada[J]. Vet Microbiol, 2013, 162(2-4):819-825.
[15] LAKKITJAROEN N, TAKAMATSU D, OKURA M, et al. Capsule loss or death:the position of mutations among capsule genes sways the destiny of Streptococcus suis[J]. FEMS Microbiol Lett, 2014, 354(1):46-54.
[16] LIU Z J, ZHENG H, GOTTSCHALK M, et al. Development of multiplex PCR assays for the identification of the 33 serotypes of Streptococcus suis[J]. PLoS One, 2013, 8(8):e72070.
[17] KERDSIN A, DEJSIRILERT S, AKEDA Y, et al. Fifteen Streptococcus suis serotypes identified by multiplex PCR[J]. J Med Microbiol, 2012, 61(Pt 12):1669-1672.
[18] KERDSIN A, AKEDA Y, HATRONGJIT R, et al. Streptococcus suis serotyping by a new multiplex PCR[J]. J Med Microbiol, 2014, 63(Pt 6):824-830.
[19] SMITH H E, VEENBERGEN V, VAN DER VELDE J, et al. The cps genes of Streptococcus suis serotypes 1, 2, and 9:development of rapid serotype-specific PCR assays[J]. J Clin Microbiol, 1999, 37(10):3146-3152.
[20] WANG K C, SUN X Q, LU C P. Development of rapid serotype-specific PCR assays for eight serotypes of Streptococcus suis[J]. J Clin Microbiol, 2012, 50(10):3329-3334.
[21] WISSELINK H J, JOOSTEN J J, SMITH H E. Multiplex PCR assays for simultaneous detection of six major serotypes and two virulence-associated phenotypes of Streptococcus suis in tonsillar specimens from pigs[J]. J Clin Microbiol, 2002, 40(8):2922-2929.
[22] BONIFAIT L, VEILLETTE M, LETOURNEAU V, et al. Detection of Streptococcus suis in bioaerosols of swine confinement buildings[J]. Appl Environ Microbiol, 2014, 80(11):3296-3304.
[23] YANG W J, CAI X H, HAO Y Q, et al. Characterization of Streptococcus suis serotype 2 blood infections using RT-qPCR to quantify glutamate dehydrogenase copy numbers[J]. J Microbiol Methods, 2010, 83(3):326-329.
[24] KING S J, LEIGH J A, HEATH P J, et al. Development of a multilocus sequence typing scheme for the pig pathogen Streptococcus suis:identification of virulent clones and potential capsular serotype exchange[J]. J Clin Microbiol, 2002, 40(10):3671-3680.
[25] OKWUMABUA O, PERSAUD J S, REDDY P G. Cloning and characterization of the gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2[J]. Clin Diagn Lab Immunol, 2001, 8(2):251-257.
[26] OKWUMABUA O, O'CONNOR M, SHULL E. A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase[J]. FEMS Microbiol Lett, 2003, 218(1):79-84.
[27] SMITH H E, DE VRIES R, VAN'T SLOT R, et al. The cps locus of Streptococcus suis serotype 2:genetic determinant for the synthesis of sialic acid[J]. Microb Pathog, 2000, 29(2):127-134.
[28] FITTIPALDI N, SEGURA M, GRENIER D, et al. Virulence factors involved in the pathogenesis of the infection caused by the swine pathogen and zoonotic agent Streptococcus suis[J]. Future Microbiol, 2012, 7(2):259-279.
[29] MARIE J, MORVAN H, BERTHELOT-HÉRAULT F, et al. Antimicrobial susceptibility of Streptococcus suis isolated from swine in France and from humans in different countries between 1996 and 2000[J]. J Antimicrob Chemother, 2002, 50(2):201-209.
[30] PADUNGTOD P, THARAVICHITKUL P, JUNYA S, et al. Incidence and presence of virulence factors of Streptococcus suis infection in slaughtered pigs from Chiang Mai, Thailand[J]. Southeast Asian J Trop Med Public Health, 2010, 41(6):1454-1461.
[31] 刘建涛,张强,宋娅静,等. 猪链球菌2型表面蛋白分支酸合成酶通过p38MAPK和NF-κB通路促进TLR4依赖的炎性反应[J]. 畜牧兽医学报, 2014, 45(11):1866-1873.
LIU J T,ZHANG Q,SONG Y J,et al. Streptococcus suis serotype 2 protein aroc induces Toll-like receptor 4-dependent inflammatory responses in RAW264.7 via p38MAPK and NF-κB signaling[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(11):1866-1873. (in Chinese)
[32] SÁNCHEZ DEL REY V, FERNÁNDEZ-GARAYZÁBAL J F, DOMÍNGUEZ L, et al. Screening of virulence-associated genes as a molecular typing method for characterization of Streptococcus suis isolates recovered from wild boars and pigs[J]. Vet J, 2016, 209:108-112.
[33] VAN CALSTEREN M R, GOYETTE-DESJARDINS G, GAGNON F, et al. Explaining the serological characteristics of Streptococcus suis serotypes 1 and 1/2 from their capsular polysaccharide structure and biosynthesis[J]. J Biol Chem, 2016, 291(16):8387-8398.
[34] OKURA M, TAKAMATSU D, MARUYAMA F, et al. Genetic analysis of capsular polysaccharide synthesis gene clusters from all serotypes of Streptococcus suis:potential mechanisms for generation of capsular variation[J]. Appl Environ Microbiol, 2013, 79(8):2796-2806.

Development of A Rapid Duplex Real-Time PCR Assay for Streptococcus suis and S. suis Serotype 2

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	HE Xiaolan, ZHAO Yankun, MENG Lu, LIU Huimin, GAO Jiaojiao, ZHENG Nan. Research Progress in Heteroresistance of Staphylococcus aureus [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1432-1445.
[2]	LI Pengxu, LI Shijing, SUN Jun, XIANG Wei, ZHAO Miaomiao, HOU Tianmu, LI Huaming, GUANG Min, CHEN Ruige, XU Mengran, WU Xiaomin, JIANG Hexiang, LEI Liancheng, ZHANG Fuxian. Molecular Subtyping and Identification of Streptococcus suis Meningitidis Type 2 and Its Biological Characteristics [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1192-1207.
[3]	Lü Ruoyi, SI Xiaohui, SUN Zhigang, SHI Xiaomin, LIU Xiaoye. Drug Resistance Situation of Streptococcus suis and Prevention Measures of Infections [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 4920-4933.
[4]	HU Xiuhua, SUN Zhixin, ZHAO Mengyang, XIE Jiaqi, WANG Min, CHEN Hailiang, GE Xin, LIU Tianlong, WANG Shaolin. Pathogenicity and Resistance Analysis of Enterococcus faecium from Wild Squirrels [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 3012-3021.
[5]	LU Wanqing, ZHAO Shasha, JIANG Songhong, TONG Zhizi, HUANG Danni, GUO Jianhua, WU Junwei, ZHOU Yang. Effect of Staphylococcus aureus on IFN-α Production in BV2 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2633-2641.
[6]	MAO Yanni, CHANG Jiawei, LI Na, WANG Xin, KANG Xinyun, MA Qiang, MA Liang, WANG Guiqin. Transcriptome Differential Expression Analysis of Staphylococcus aureus in Biofilm State and Planktonic State [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2697-2707.
[7]	DU Yu, PU Xiaofeng, CHEN Xiaomeng, Lü Fenfen, WANG Li, ZHANG Zehua, ZHANG Baojiang, SU Yan. Identification and Evolutionary Analysis of Three Novel SeM Genotypes of Donkey Streptococcus equi subsp. equi Isolates in Xinjiang [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 231-240.
[8]	ZHANG Haixia, ZHANG Zhenbiao, DAI Hegen, SONG Yu, LI Lei, XIA Zhaofei. Analysis of Epidemiological Characteristics of Clinical Staphylococcus pseudintermedius Isolated from Dogs [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2561-2568.
[9]	ZHANG Jing, CAI Ping, HE Yizhuo, CHEN Weitao, HUANG Yuchen, JIANG Hongxia. Prevalence of Antimicrobial Resistance and Virulence Factors in Enterococci from Poultry Farms in Guangdong Province [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2650-2659.
[10]	ZHANG Jinning, QIAN Mengying, TANG Yongjie, MI Siyuan, SHI Kerong, YU Ying. The Adhesion Effect of Staphylococcus aureus Surface Protein A on Dairy Cattle Mammary Epithelial Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1369-1377.
[11]	LÜ Fenfen, MA Xiaohui, WANG Li, MA Chen, ZHANG Baojiang, SU Yan. Immunogenicity Analysis of Three Different Antigen Proteins of Streptococcus equi in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(3): 752-762.
[12]	ZHANG Hang, LI Xinpu, WU Xiaohu, DING Xuezhi, YAN Zuoting, WANG Shengyi, LI Hongsheng. Investigation of Biofilm Formation, Virulence Genes and agr Typing of Staphylococcus aureus from Bovine Mastitis Cases [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11): 2903-2910.
[13]	MA Qiang, YANG Rui, WAN Jiahong, CHANG Jiawei, WEI Yanqin, WANG Guiqin. Study on β-lactamase and Its Action Mode of Staphylococcus aureus Isolated from Cows in Ningxia [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 1138-1148.
[14]	MA Qiang, WANG Yihui, CHANG Jiawei, WAN Jiahong, WEI Yanqin, WANG Guiqin. The Relationship between Cell Wall Thickening of Staphylococcus aureus and Resistance to β-lactams [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 602-611.
[15]	MA Weiwu, ZHOU Xuezhang. Expression of Panton-Valenine Leukocidin from Staphylococcus aureus and Its Effects on Bovine Mammary Epithelial Cells [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(10): 2105-2112.