炭疽杆菌双重荧光定量PCR检测技术的建立和应用

doi:10.11843/j.issn.0366-6964.2019.08.015

Abstract

Abstract: In order to detect Bacillus anthracis rapidly and accurately, two sets of specific primers and TaqMan probes were designed according to the chromosomal sequence BA5345 gene and virulent plasmid sequence PA gene to establish a duplex real-time PCR assay for detection of Bacillus anthracis. The specificity, sensitivity and repeatability were tested, respectively. Then the duplex real-time PCR was used to detect clinical samples and compared with conventional PCR. Results showed that the Bacillus anthracis could be identified specifically and without any cross reaction with other bacillus of Bacillus cereus group. Besides, the detection limits were 8.0 and 7.8 copies·μL^-1 for BA5345 gene and PA gene, respectively. The coefficients of variations were less than 1.5% for both intra-assay and inter-assay. Thirty-five samples contaminated by Bacillus anthracis were detected by the established assay, BA5345 positive rate was 80.0% and PA positive rate was 82.9%. The sensitivity of duplex real-time PCR was significantly higher than that of conventional PCR. The results indicate that the detection assay could be applied for rapid and high through-put detection of Bacillus anthracis.

CLC Number:

S852.616

WANG Suhua, SHUAI Jiangbing, LI Zhou, YUAN Shuhui, WU Shaoqiang, Lü Jizhou, ZHAO Zhiguo. Establishment and Application of a Duplex Real-time PCR Assay for Detection of Bacillus anthracis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(8): 1658-1665.

0
/ / Recommend

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

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2019.08.015

https://www.xmsyxb.com/EN/Y2019/V50/I8/1658

References

[1] OLSEN J S, SKOGAN G, FYKSE E M, et al. Genetic distribution of 295Bacillus cereus group members based on adk-screening in combination with MLST (Multilocus Sequence Typing) used for validating a primer targeting a chromosomal locus in B. anthracis[J]. J Microbiol Methods, 2007, 71(3):265-274.
[2] KENAR L, ORTATATLI M, KARAYILANOGLU T, et al. Comparison of real-time polymerase chain reaction and conventional polymerase chain reaction methods for the rapid identification of Bacillus anthracis[J]. Mil Med, 2007, 172(7):773-776.
[3] ANTWERPEN M H, ZIMMERMANN P, BEWLEY K, et al. Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis[J]. Mol Cell Probes, 2008, 22(5-6):313-315.
[4] HADJINICOLAOU A V, DEMETRIOU V L, HEZKA J, et al. Use of molecular beacons and multi-allelic real-time PCR for detection of and discrimination between virulent Bacillus anthracis and other Bacillus isolates[J]. J Microbiol Methods, 2009, 78(1):45-53.
[5] STRAUB T, BAIRD C, BARTHOLOMEW R A, et al. Estimated copy number of Bacillus anthracis plasmids pXO1 and pXO2 using digital PCR[J]. J Microbiol Methods, 2013, 92(1):9-10.
[6] KOLSTØ A B, TOURASSE N J, ØKSTAD O A. What sets Bacillus anthracis apart from other bacillus species?[J]. Annu Rev Microbiol, 2009, 63:451-476.
[7] 李文涓, 石一,李沈玲,等. 炭疽与蜡样芽胞杆菌rpoB基因交叉反应的分子分析[J]. 现代预防医学, 2017, 44(12):2238-2244.
LI W J, SHI Y, LI S L, et al. Molecular analysis on cross-reaction of rpoB gene between Bacillus anthracis and Bacillus cereus[J]. Modern Preventive Medicine, 2017, 44(12):2238-2244. (in Chinese)
[8] CIESLIK P, KNAP J, KOLODZIEJ M, et al. Real-time PCR identification of unique Bacillus anthracis sequences[J]. Folia Biol, 2015, 61(5):178-183.
[9] HU X M, HANSEN B M, HENDRIKSEN N B, et al. Detection and phylogenic analysis of one anthrax virulence plasmid pXO1 conservative open reading frame ubiquitous presented within Bacillus cereus group strains[J]. Biochem Biophys Res Commun, 2006, 349(4):1214-1219.
[10] DERZELLE S, GIRAULT G, ROEST H I J, et al. Molecular diversity of Bacillus anthracis in the Netherlands:investigating the relationship to the worldwide population using whole-genome SNP discovery[J]. Infect Genet Evol, 2015, 32:370-376.
[11] ROULI L, MBENGUE M, ROBERT C, et al. Genomic analysis of three African strains of Bacillus anthracis demonstrates that they are part of the clonal expansion of an exclusively pathogenic bacterium[J]. New Microbes New Infect, 2014, 2(6):161-169.
[12] PANNUCCI J, OKINAKA R T, SABIN R, et al. Bacillus anthracis pXO1 plasmid sequence conservation among closely related bacterial species[J]. J Bacteriol, 2002, 184(1):134-141.
[13] RADNEDGE L, AGRON P G, HILL K K, et al. Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis[J]. Appl Environ Microbiol, 2003, 69(5):2755-2764.
[14] IRENGE L M, GALA J L. Rapid detection methods for Bacillus anthracis in environmental samples:a review[J]. Appl Microbiol Biotechnol, 2012, 93(4):1411-1422.
[15] ÅGREN J, HAMIDJAJA R A, HANSEN T, et al. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences[J]. Virulence, 2013, 4(8):671-685.
[16] BODE E, HURTLE W, NORWOOD D. Real-time PCR assay for a unique chromosomal sequence of Bacillus anthracis[J]. J Clin Microbiol, 2004, 42(12):5825-5831.
[17] 谭维国, 陈文琦,吕恒,等. 炭疽杆菌双重实时荧光PCR检测方法的建立[J]. 东南国防医药, 2013, 15(6):556-559.
TAN W G, CHEN W Q, LV H, et al. Development of a duplex real-time PCR assay for detection of Bacillus anthracis[J]. Military Medical Journal of Southeast China, 2013, 15(6):556-559. (in Chinese)
[18] 刘东立, 李文涓,石一,等. TaqMan-LNA探针多重实时荧光定量PCR检测炭疽芽胞杆菌的研究[J]. 中国病原生物学杂志, 2018, 13(3):244-248, 258.
LIU D L, LI W J, SHI Y, et al. A study on use of TaqMan-LNA probe multiple real-time fluorescence quantitative PCR to detect Bacillus anthracis[J]. Journal of Pathogen Biology, 2018, 13(3):244-248, 258. (in Chinese)

Establishment and Application of a Duplex Real-time PCR Assay for Detection of Bacillus anthracis

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 7

Recommended Articles

Metrics

Comments

[1]	ZENG Chengrong, WANG Na, BI Wenwen, MEI Shihui, HE Guangxia, ZHANG Junjie, CHEN Ze, WEN Ming, ZHOU Bijun. Metabonomics Analysis of Duck Ileum Infected by Clostridium perfringens Type A [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2555-2569.
[2]	WU Ke, FENG Hang, WANG Juan, YANG Zengqi. Whole Genome Sequencing and Molecular Characterization Analysis of Clostridium perfringens Type D Strains from Guanzhong Dairy Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3967-3974.
[3]	HUANG Xiaoyu, YANG Qiaoli, YAN Zunqiang, WANG Pengfei, SHI Hairen, GUN Shuangbao. Characterization of circRNA Expression Profiles Involved in Intestines of Clostridium Perfringens Type C-infected Diarrheal Piglet [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4058-4070.
[4]	ZANG Jianghua, AN Yina, WANG Jing, WANG Kezhi, YANG Jingjing, GAO Min, FENG Landi, TAN Shuyu, HU Yanxin, DONG Yanjun. Investigation on Pathogenicity and Drug Efficacy of Clostridium perfringens Type A [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3561-3569.
[5]	ZHANG Siyu, WANG Yujiong, ZENG Jin. Transcriptome Analysis of Intestinal Injury Induced by Clostridium perfringens Type C Exotoxin in Mouse [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3570-3581.
[6]	DU Ji-ge, ZHU Zhen, XUE Qi, LI Qi-hong, YIN Chun-sheng, PENG Xiao-bing, YAO Wen-sheng, KANG Kai, CHEN Xiao-yun. Evaluation of Protective Efficacy of Recombinant Mutant of Clostridium perfringens ε Toxin [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 777-785.
[7]	WANG Ying-ying，QIAO Yi-ran，ZHAO Lei，LIU Peng，REN Yu-dong，LI Guang-xing，HUANG Xiao-dan，ZHANG Rui-li，YANG Gui-jun. The Study on Cytotoxicity of Recombinant NetB Toxin of Clostridium perfringens [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(5): 1018-1025.