牛羊猪种布鲁氏菌快速鉴别PCR方法的建立及评价
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摘要
目的建立一种可在一个反应体系中同时鉴别布鲁氏菌及牛羊猪种布鲁氏菌的快速PCR鉴别方法。方法将布鲁氏菌及牛羊猪种布鲁氏菌的扩增引物进行优化组合,建立全新的BAMS-PCR方法;随后对该方法的特异性和灵敏度进行评价,并对现场分离的219株布鲁氏菌进行鉴别,评价其在布鲁氏菌鉴别中的应用价值。最后,用该方法对临床标本中布鲁氏菌的DNA进行扩增,评价其在临床诊断中的实用性。结果 BAMS-PCR方法可在同一反应中同时鉴别布鲁氏菌及牛种(1,2,4型),羊种(1,2和3型)和猪种(1型)布鲁氏菌,并有较好的特异性和敏感性。布鲁氏菌属,牛种,猪种和羊种布鲁氏菌引物的检测灵敏度分别为10 pg/μL,100 pg/μL, 10 pg/μL和100 pg/μL。BAMS-PCR对219株临床分离菌株的鉴定结果和常规生物分型方法的鉴定符合率为100%。经BAMS-PCR检测,97份临床血清样本仅6份为阳性,全血和组织(羊脾)样本全部为阴性。结论 BAMS-PCR是一种简便、快速、高效、准确的布鲁氏菌鉴别方法,可作为临床分离菌株的首选鉴别方法。
The aim of this study is to establish a rapid PCR discriminate assay for simultaneous identified Brucella genus, B. abortus, B. melitensis and B. suis in single reaction system. Amplification primers of Brucella genus and B. abortus, B. melitensis, and B. suis were combined and optimizated, a kind of novelty BAMS-PCR was established. Subsequently, specificity and sensitivity of BAMS-PCR were evaluated. In present study, 219 Brucella spp. were identified for assessed practicable of BAMS-PCR. Finally, DNA of Brucella clinical sample were detected by BAMS-PCR for evaluated using valuable in clinical diagnosis of Brucellosis. BAMS-PCR can simultaneous identified Brucella genus, B. abortus(bv.1,2 and 4), B. melitensis(bv.1-3) and B. suis(bv.1) in single reaction system, and this method had very higher specificity and sensitivity, detecting sensitivity of primers of Brucella genus, B. abortus, B. melitensis and B. suis with 10 pg/μL,100 pg/μL, 10 pg/μL and 100 pg/μL, respectively. There were 100% identification coincidence rate between BAMS-PCR and convention biotypes testing based on amplificated results of 219 Brucella spp. BAMS-PCR detected showing that there were only 6 serum samples were positive, others(whole blood and tissues(sheep spleen)) samples were all negative. BAMS-PCR was a simple, rapid, high efficiency and accuracy identification assay for Bruclla spp., could as first choice discriminate method of clinical isolates Bruclla spp.
The aim of this study is to establish a rapid PCR discriminate assay for simultaneous identified Brucella genus, B. abortus, B. melitensis and B. suis in single reaction system. Amplification primers of Brucella genus and B. abortus, B. melitensis, and B. suis were combined and optimizated, a kind of novelty BAMS-PCR was established. Subsequently, specificity and sensitivity of BAMS-PCR were evaluated. In present study, 219 Brucella spp. were identified for assessed practicable of BAMS-PCR. Finally, DNA of Brucella clinical sample were detected by BAMS-PCR for evaluated using valuable in clinical diagnosis of Brucellosis. BAMS-PCR can simultaneous identified Brucella genus, B. abortus(bv.1,2 and 4), B. melitensis(bv.1-3) and B. suis(bv.1) in single reaction system, and this method had very higher specificity and sensitivity, detecting sensitivity of primers of Brucella genus, B. abortus, B. melitensis and B. suis with 10 pg/μL,100 pg/μL, 10 pg/μL and 100 pg/μL, respectively. There were 100% identification coincidence rate between BAMS-PCR and convention biotypes testing based on amplificated results of 219 Brucella spp. BAMS-PCR detected showing that there were only 6 serum samples were positive, others(whole blood and tissues(sheep spleen)) samples were all negative. BAMS-PCR was a simple, rapid, high efficiency and accuracy identification assay for Bruclla spp., could as first choice discriminate method of clinical isolates Bruclla spp.
引文
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[14] Mitka S,Anetakis C,Souliou E,et al.Evaluation of different PCR assays for early detection of acute and relapsing brucellosis in humans in comparison with conventional methods[J].J Clin Microbiol,2007,45 (4):1211-1218.DOI:10.1128/JCM.00010-06
[15] Gwida M,El-Ashker M,Melzer F,et al.Use of serology and real time PCR to control an outbreak of bovine brucellosis at a dairy cattle farm in the Nile Delta region,Egypt[J].Ir Vet J,2015,69:3.DOI:10.1186/s13620-016-0062-9
[2] Olivares R,Vidal P,Sotomayor C.,et al.Brucellosis in chile:description of a series of 13 cases[J].Rev Chilena Infectol,2017,34 (3):243-247.DOI:10.4067/S0716-10182017000300 006
[3] Serpa JA,Knights S,Farmakiotis D,et al.Brucellosis in adults and children:a 10-year case series at two large academic hospitals in houston,Texas[J].South Med J,2018,111 (6):324-327.DOI:10.14423/SMJ.0000000000000810
[4] Ntirandekura JB,Matemba LE,Kimera S,et al.Association of Brucellosis with abortion prevalence in humans and animals in africa:a review[J].Afr J Reprod Health,2018,22 (3):120-136.DOI:10.29063/ajrh2018/v22i3.13
[5] Zakaria AM,Ahmed SF,Motawae MS.Seropositivity in animals and risk of occupational brucellosis among abattoirs personnel associated with poor work practices and absence of safety policy in Egypt[J].Int J Occup Environ Health,2018,24(1/2):55-60.DOI:10.1080/10773525.2018.1516839
[6] Mukherjee F,Jain J,Patel V,et al.Multiple genus-specific markers in PCR assays improve the specificity and sensitivity of diagnosis of brucellosis in field animals[J].J Med Microbiol,2007,56 (Pt 10):1309-1316.DOI:10.1099/jmm.0.47160-0
[7] Navarro E,Fernandez JA,Escribano J,et al.PCR assay for diagnosis of human brucellosis[J].J Clin Microbiol,1999,37 (5):1654-1655.
[8] Gemechu MY,Gill JP,Arora AK,et al.Polymerase chain reaction (PCR) assay for rapid diagnosis and its role in prevention of human brucellosis in Punjab,India[J].Int J Prev Med,2011,2 (3):170-177.
[9] Kumar S,Tuteja U,Sarika K,et al.Rapid multiplex PCR assay for the simultaneous detection of the Brucella Genus,B.abortus,B.melitensis,and B.suis[J].J Microbiol Biotechnol,2011,21 (1):89-92.DOI:10.4014/jmb.1007.07051
[10] Kamal IH,Al Gashgari B,Moselhy SS,et al.Two-stage PCR assay for detection of human brucellosis in endemic areas[J].BMC Infect Dis,2013,13:145.DOI:10.1186/1471-2334-13-145
[11] Navarro E,Casao MA,Solera J,Diagnosis of human brucellosis using PCR[J].Expert Rev Mol Diagn,2004,4 (1):115-123.DOI:10.1586/14737159.4.1.115
[12] Batinga MCA,de Lima JTR,Gregori F,et al.Comparative application of IS711-based polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for canine brucellosis diagnosis[J].Mol Cell Probes,2018,39:1-6.DOI:10.1016/j.mcp.2018.02.003
[13] Shang DQ,Xiao DL,Yin JM.Epidemiology and control of brucellosis in China[J].Vet Microbiol,2002,90 (1-4):165-182.DOI:10.1016/S0378-1135(02)00252-3
[14] Mitka S,Anetakis C,Souliou E,et al.Evaluation of different PCR assays for early detection of acute and relapsing brucellosis in humans in comparison with conventional methods[J].J Clin Microbiol,2007,45 (4):1211-1218.DOI:10.1128/JCM.00010-06
[15] Gwida M,El-Ashker M,Melzer F,et al.Use of serology and real time PCR to control an outbreak of bovine brucellosis at a dairy cattle farm in the Nile Delta region,Egypt[J].Ir Vet J,2015,69:3.DOI:10.1186/s13620-016-0062-9