Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus,hepatitis B virus,and human immunodeficiency virus 1

BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Efficacy of stool multiplex polymerase chain reaction assay in adult patients with acute infectious diarrhea

BACKGROUND Recently,stool multiplex polymerase chain reaction(PCR)tests have been developed for identifying diarrhea-causing bacterial pathogens.Furthermore,fecal calprotectin is a well-known effective marker for intestinal mucosal inflammation.AIM To evaluate the efficacy of stool multiplex PCR and fecal calprotectin in acute infectious diarrhea.METHODS Overall,400 patients with acute infectious diarrhea were enrolled from Kangdong Sacred Heart Hospital(January 2016 to December 2018).Multiplex PCR detected 7 enteropathogenic bacteria including Salmonella,Campylobacter,Shigella,Escherichia coli O157:H7,Aeromonas,Vibrio,and Clostridium difficile.We reviewed clinical and laboratory findings using stool multiplex PCR.RESULTS Stool multiplex PCR test detected considerably more bacterial pathogens than stool culture(49.2%vs 5.2%),with Campylobacter as the most common pathogen(54%).Patients with positive stool PCR showed elevated fecal calprotectin expression compared to patients with negative stool PCR(1124.5±816.9 mg/kg vs 609±713.2 mg/kg,P=0.001).C-reactive protein(OR=1.01,95%CI:1.001-1.027,P=0.034)and sigmoidoscopy-detected colitis(OR=4.76,95%CI:1.101-20.551,P=0.037)were independent factors in stool PCR-based detection of bacterial pathogens.Sensitivity and specificity of calprotectin were evaluated to be 70.5%and 60.9%,respectively(adjusted cut-off value=388 mg/kg).CONCLUSION Stool multiplex PCR test has increased sensitivity in detecting pathogens than conventional culture,and it is correlated with calprotectin expression.Stool multiplex PCR and calprotectin may be effective in predicting clinical severity of infectious diarrhea.

Human papillomavirus 16 physical status detection in preinvasive and invasive cervical carcinoma by multiplex real-time polymerase chain reaction

Objective: To explore an ideal approach for detecting the physical status of HPV-16 in clinic use and to investigate the integrated HPV-16 in CINs and cervical cancer. Methods: Multiplex real-time PCR method was established to quantify the copy numbers of E2 and E6 genes (E2/E6) for analysis of the physical status of HPV-16 DNA and this assay was compared to Southern blot analysis. HPV-16-containing paraffin-embedded tissues including 49 CINs and 51 cervical squamous cancers were detected using the method. Results: (1) The cutoff ratio of E2/E6 to distinguish pure episomal from mixed HPV-16, was 0.81 in the multiplex real-time PCR; (2) The agreement rate between multiplex real-time PCR and Southern blot was 81.5% (the Kappa statistic was 0.844, P<0.001); (3) HPV-16 DNA existed in an episomal form in 57.1% and mixed form in 42.9% of CIN I lesions; The concomitant form of HPV-16 (>70%) constituted the majority in CIN II and CIN III; HPV-16 DNA mostly integrated into the host chromosome (s) in squamous cervical cancers (68.6%); (4) The incidence of HPV-16 integration was increased with the degree of cervical lesions; (5) The frequency of pure integrated HPV-16 in stage II+III (88%) was signifi- cantly higher than that in stage I (33.3%). Conclusion: (1) Mutiplex real-time PCR provides a rapid, sensitive and reliable method for clinic detection of the physical state of HPV-16 DNA; (2) The integration of the HPV-16 DNA is a very early and important event in the progression from preinvasive to invasive cervical cancer; (3) The pure integrated status of HPV-16 in cervical cancer may be associated with poor prognosis of cervical cancer, but further study will be needed to prove its prog- nostic significance.

DETECTION OF PATHOGENS CAUSING GENITAL ULCER DISEASE BY MULTIPLEX POLYMERASE CHAIN REACTION

Objective To establish a multiplex polymerase chain reaction (M-PCR) assay for simultaneous detection of pathogens causing genital ulcer disease (GUD). Methods Based on the gene-specific region of the following pathogens: Chlamydia trachomatis omp1/ompb, herpes simplex virus (HSV) DNA polymerase, Treponema pallidum tpp47, Haemophilus ducreyi 16s rRNA, four sets of primers were designed and an M-PCR assay was developed to detect four pathogens in one test. The assay was evaluated with diagnostic result of golden standard for each pathogen. Results Of the 51 clinical samples, M-PCR showed slightly higher positive rate (47.1%) of HSV than cell culture (23.6%).Meanwhile, the positive rate of T. pallidum detected by M-PCR and dark-field microscopy was 19.6% (10/51) and 15.7% (8/51), respectively. Only one sample was positive for H. ducreyi and no sample was positive for C. trachomatis detected by both M-PCR assay and culture. Conclusion This primary study indicated that M-PCR assay can simultaneously and rapidly detect the four etiologic pathogens causing GUD.

利用mPCR方法检测巴氏奶中致病菌的鲁棒性研究

为解决巴氏奶货架期短与致病菌传统检测方法耗时长相矛盾问题,建立一种检测巴氏奶中的阪崎克罗诺杆菌、大肠杆菌和沙门氏菌的鲁棒性多重聚合酶链式反应(multiple polymerase chain reaction,mPCR)方法。旨在常规mPCR的基础上深入研究,提高方法的准确性和稳定性。首先,针对每个目标菌株选择两种基因,并设计两组特异性引物,建立两套mPCR扩增体系,进行双重检测;然后,在不改变方法灵敏度的前提下,对优化了的退火温度进行范围稳定性选择;最后,将提出方法与国标方法 GB 4789.40-2016、GB 4789.38-2012、GB 4789.4-2016进行比较,同时检测人工污染样品并评价应用效果。结果表明,第一套mPCR检测巴氏奶中三种致病菌可在退火温度59~59.5℃(温差0.5℃)的范围内,具有较好的检测准确性和稳定性,灵敏度为10 CFU/mL。第二套mPCR检测巴氏奶中三种致病菌可在退火温度57.5~58.5℃(温差1℃)的范围内,不影响方法的准确性和稳定性,灵敏度为10 CFU/mL。采用建立的鲁棒性mPCR方法对人工污染的50份巴氏奶样品进行检测,检测结果与国标方法一致。在检测时效上需要4 h,较国标方法(检测时间为62~148 h)显著(P<0.05)缩短检测时间。该方法对微生物流行病学调查和研究,尤其是对巴氏奶中致病菌的快速检测和安全控制具有一定的实际应用价值。

Clinical Application of Multiplex PCR Assay for the Diagnosis of the Etiology of Genital Ulcer Disease Among Patients Attending STD Clinics in Guangzhou, China

Objectives: To develop a method of simultaneous PCRdetection of Haemophilus ducreyi, Treponema pallidum, andHerpes Simplex Virus Types 1 and 2 from genital ulcersamong patients attending STD clinics in Guangzhou, China;and evaluate the clinical application of multiplex PCR (M-PCR) assay for diagnosing the etiology of genital ulcerdiseases (GUD). Methods: 244 patients with a genital ulcer were evaluated.Clinical etiology of GUD was based on physical appearanceand microbiologic evaluations that included dark fieldmicroscopy examination (D-F) and serology test for syphilis(STS). Swabs of each genital ulcer were tested for HSVantigen by enzyme immunoassay (EIA) and processed in anM-PCR assay for simultaneous detection of T. pallidum, HSVand H. ducreyi. Results: The standard strains of T. pallidum, HSV and H.ducreyi were amplified by M-PCR, producing amplifiedproducts of 260bp,432bp,170bp, respectively. The sensitivityof M-PCR is 102pg DNA. M-PCR assay for T. pallidum, HSVand H. ducreyi showed good agreement when compared withD-F detection for T. pallidum, STS, H. ducreyi culture and EIAfor HSV antigen (Kappa scores are 0.774,0.704,0.793,0.756,respectively). Conclusions: The M-PCR is a convenient, accurate andreliable assay for the detection of T. pallidum, HSV and H.ducreyi from genital ulcers, and can be used as a method of diagnosing the etiology of GUD.

4种植物源性成分多重real-time PCR检测方法的建立及其在食用淀粉中的应用

建立一种可同时快速检测红薯、木薯、马铃薯、玉米源性成分的多重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以红薯g3pdh基因、木薯g3pdh基因、马铃薯UGPase基因、玉米zSSIIb基因为靶基因设计特异性引物和TaqMan探针,以18S rRNA基因为内参基因,建立多重real-time PCR方法,开展方法学验证,并对不同掺入比例模拟样品和实际淀粉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。与15种非目标源性均无交叉反应;对目标DNA的检测灵敏度可达到3×10^(-3) ng/μL,且具有良好的线性关系和扩增效率;对淀粉样品的检出限可达0.1%,对50份实际样品进行检测,结果与参比方法一致,说明建立的多重real-time PCR法可用于食用淀粉种类掺假鉴别检测。

多重PCR毛细管电泳细菌快速鉴定方法的建立和临床应用研究

目的针对引起人类感染的常见病原菌建立一种基于多重PCR毛细管电泳技术的快速细菌鉴定方法,评估其临床应用价值。方法建立23种常见病原菌的多重PCR毛细管电泳检测体系。收集150例临床微生物检测标本,分别用多重PCR毛细管电泳法(以下简称多重PCR法)、培养法进行检测。对两种方法的检测结果进行比较,评价多重PCR法的检测效能。结果150例标本中多重PCR法检出15种病原菌、培养法检出14种病原菌。多重PCR法检测阳性率为73.3%,培养法为70.0%,差异无统计学意义(P>0.05)。多重PCR法对肺炎链球菌的检出率为16.0%,培养法为6.0%,差异有统计学意义(P<0.05)。多重PCR法对混合菌标本的检出率为15.3%,培养法未检出混合菌标本。多重PCR法与培养法检测结果的符合率为79.3%。多重PCR法检测时间为3~6 h,培养法为2~4 d。结论与培养法比较,多重PCR法具有较高的时效性,对肺炎链球菌及混合菌标本具有较高的检出率,可满足临床标本病原微生物快速初筛的需求。

Diagnosis of the accurate genotype of HKαα carriers in patients with thalassemia using multiplex ligation-dependent probe amplification combined with nested polymerase chain reaction

Background:Patients carrying the HongKongαα(HKαα)allele and-α3.7/αααanti-4.2 could be misdiagnosed as-α3.7/ααby the current conventional thalassemia detection methods,leading to inaccurate genetic counseling and an incorrect prenatal diagnosis.This study was aimed to accurately analyze the genotypes of HKααcarriers and-α3.7/αααanti-4.2.Methods::Samples were collected in our hospital from July 2017 to October 2019.Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction(Gap-PCR)and reverse dot blot(RDB).Anti-4.2 multiplex-PCR was used to confirm carriers of theαααanti-4.2 duplication with-α3.7 deletion.Two-round nested PCR and multiplex ligation-dependent probe amplification(MLPA)were applied to accurately identify and confirm their genotypes.For data analysis,we used descriptive statistics and Fisher’s exact tests.Results::Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples.The results showed thatα,β,andαβcompound thalassemia were identified in 1190(46.78%),1286(50.55%),and 68(2.67%)cases,respectively.A total of 227 samples from thalassemia patients were identified as-α3.7/ααby Gap-PCR,and the genotypes of two samples were uncertain.There was a difference between Gap-PCR and combined groups(Gap-PCR combined with nested PCR and MLPA)in detecting HKαα(P<0.05).Among the 229 patients,20 patients were identified as HKααcarriers and one was identified as-α3.7/ααα anti-4.2 by two-round nested PCR and MLPA,including 15 patients with HKαα/αα,three with HKαα/αα and β-thalassemia coinheritance,one with HKαα/-SEA,one with HKαα/-α4.2 andβ-thalassemia coinheritance,and one with-α3.7/αααanti-4.2 and β-thalassemia coinheritance.Conclusions::αααanti-4.2 and HKααgenotypes of patients carrying-α3.7 need to be detected to reduce the misdiagnosis rate of patients carrying HKααand-α3.7/αααanti-4.2 alleles.More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.

Rapid prenatal diagnosis of trisomy 21 by fluorescent quantitative multiplex polymerase chain reaction

Trisomy 21, also named Down syndrome was the most frequent autosomal aneuploidy and the most common cause of mental retardation. Fifty percent patients had congenital heart malformation. Every 20 minutes one case of trisomy 21 was born, and the incidence rate was 1 in 600 to 800 newborns in China.1 In two thirds of cases with trisomy 21, there was a spontaneous abortion, so the actual incidence was higher than that obtained postnatally.

MPCR检测食品中大肠杆菌O157和单核增生李斯特氏菌的研究

根据大肠杆菌O157(Escherichia coliO157,E.coliO157)的志贺样毒素基因slt和"黏附抹平"因子eaeA基因、单核增生李斯特氏菌(Listeria monocytohenes,LM)的编码溶血素O的hlyA基因和毒力基因plcA,分别设计上游和下游的slt、eaeA、hlyA和plcA四对引物,对应扩增片段依次为780、450、708、600bp。通过对单管多重PCR(multiplex PCR,MPCR)扩增的特异性、敏感性分析以及对单管多重PCR扩增条件,如Mg2+浓度、dNTPs浓度和退火温度等的优化,建立快速检测大肠杆菌O157和单增李斯特菌的多重PCR方法,该方法能同时检测到0.50ng的E.coliO157和单增李斯特菌基因组DNA,并且操作简便、快速,具有良好的敏感性和特异性,能够快速实现对食品中多种致病菌的诊断和监控。

Forensic Identification of Four Indian Snake Species Using Single Multiplex Polymerase Chain Reaction

Among different endangered animal species,snakes are the most neglected creature looked at with apathy and therefore,are ruthlessly killed,illegally trafficked,and poached for their venom,lucrative skin,meat,and bones for manufacturing of medicines,accessories,and food items.Establishing the identity of the endangered snake species is important for punishing the offenders under Wildlife Protection Act(WPA)(1972)but morphological characters fail to establish identity as they are often altered.The technique of identification of snake species at molecular level holds very effective conclusion in punishing offender.Here,we have constructed and demonstrated a novel multiplexing polymerase chain reaction technique,using 16S rRNA and C-mos gene for identification of four Indian snake species,namely Ptyas mucosa,Daboia russellii,Naja naja,and Xenochrophis piscator.They are listed in Appendix-II and III of convention on international trade in endangered species of wild fauna and flora and Schedule II;Part II of Indian WPA,1972.Therefore,it may be considered a functional tool for establishing species-specific identity of four Indian snake species and promising to be useful for their conservation.

VALUE OF POLYMERASE CHAIN REACTION ASSAY IN DIAGNOSIS OF INTESTINAL TUBERCULOSIS AND DIFFERENTIATION FROM CROHN'S DISEASE

It is difficult to make a precise diagnosis of intestinal tuberculosis and to differentiate it from Crohn's disease. For evaluating Polymerase Chain Reaction (PCR) assay in these two aspects, 36 specimens of intestinal tuberculosis from surgical resections and endoscopic biopsies and 26 Crohn's disease samples were subjected to PCR assay. 21 specimens of normal colon tissue surrounding cancer were used as the control. Oligonucleotides derived from the IS 6110 sequence, which is repeated in M. tuberculosis chromosome and highly specific for the M. tuberculosis complex, were used as a primer. The amplified PCR products were detected by examination of ethidium-bromide-stained polyacrylamide gels. The specificity of PCR products was confirmed by digestion with Sal 1 restrictive endonuclease and southern blot hybridization using digoxigenin-labeled probe. The results showed that the M. tuberculosis DNA was identified in 27 / 36 intestinal tuberculosis, but none of 26 Crohn's disease. Acid fast bacilli were only found in 16 / 36 intestinal tuberculosis. In conclusion, as a rapid, sensitive, and specific pathogenic method in diagnosis of intestinal tuberculosis, PCR assay has been developed in this study, and is considered valuable in the differentiation between intestinal tuberculosis and Crohn's disease.

多重实时荧光定量聚合酶链式反应在芝麻酱掺假鉴别中的应用

目的研究基于分子生物学技术,建立多重实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)技术检测芝麻酱中的植物源性成分,实现芝麻酱的快速掺假鉴别,突破单标准单物种检测的局限性。方法该研究以芝麻2S albumim mRNA基因、花生Ara b2基因、大豆Lectin基因、玉米adh1基因设计特异性引物和探针,优化反应体系,建立多重实时荧光定量PCR技术检测芝麻酱中的芝麻源性成分、花生源性成分、大豆源性成分、玉米源性成分,并应用于市售的芝麻酱样本检测分析。结果该方法高效低成本,特异性好,对小米、绿豆等10种非目标源性成分无特异性扩增;对芝麻源性成分、花生源性成分、大豆源性成分、玉米源性成分的最低检出限为100 pg/μL,具有较高的灵敏度;应用建立的方法,对市售21份芝麻酱样本进行检测分析,检测结果与标签标注不符合的有4份,标签标注符合率为80.95%,与参比方法检测结果一致。结论建立的多重实时荧光定量PCR技术对加工成品的检测具有较好的适用性,为芝麻酱的掺假鉴别提供了一种新的分子生物学方法。

市售食品21种动植物过敏原成分的检测分析

为进一步明确市售食品动植物过敏原标注情况,该研究建立一种可同时检测21种动植物过敏原成分的多重连接依赖性探针扩增(MLPA)技术,并对其进行灵敏度实验,并结合实时荧光定量聚合酶链式反应(RT-fqPCR)检测技术对38种市售预包装食品中动植物过敏原成分进行比较分析。结果表明,采用MLPA检测技术可同时对21种动植物过敏原成分进行检测,扩增峰之间不存在交叉干扰,扩增峰实际大小和理论大小相差≤3 bp,检出最低脱氧核糖核酸(DNA)质量浓度为1 ng/μL;RT-fqPCR、MLPA技术检测标注过敏原成分食品的检出率分别为51.5%、44.1%,此外,MLPA检测法检出了6个标注可能含有过敏原成分样品中的过敏原成分,10个样品中未标记的过敏原成分。因此,采用MLPA技术检测21种动植物过敏原成分的灵敏度更高。

Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients

AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P< 0.001) and preoperative serum levels of CEA (P=0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented signif icant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RTPCR assay can provide useful information concerning disease stage and overall survival of CRC patients.

Dual-priming oligonucleotide-based multiplex PCR using tissue samples in rapid urease test in the detection of Helicobacter pylori infection

AIM:To investigate whether tissue samples processed by the rapid urease test(RUT)kit are suitable for dualpriming oligonucleotide-based multiplex polymerase chain reaction(DPO-PCR)to detect Helicobacter pylori(H.pylori).METHODS:A total of 54 patients with specific gastrointestinal symptom were enrolled in this study.During endoscopy,gastric biopsy specimens were taken for histology,RUT,and DPO-PCR.DPO-PCR was performed on gastric biopsy samples and tissue samples that were analyzed by RUT at 2 separate institutes.In detecting H.pylori,the concordance rate of the DPO-PCR tests between the tissue samples that had been submitted to RUT and the gastric biopsy samples was investigated.RESULTS:H.pylori co-occurred with 76.0%(19/25)of gastric ulcers,64.3%(9/14)of duodenal ulcers,and 33.3%(4/12)of gastritis cases.H.pylori infection was found in 100%(3/3)of the patients with both gastric and duodenal ulcers.Overall,H.pylori was detected in 35 of 54(64.8%)patients.The diagnostic sensitivities of histology,RUT,and DPO-PCR were85.7%(30/35),74.3%(26/35),and 97.1%(34/35),respectively(P=0.02).The positive predictive value(PPV)of DPO-PCR was 94.4%,whereas the negative predictive value(NPV)was 94.7%.In the rapid urease test(CLOtest)-negative cases,the frequency of positive DPO-PCR and histologic results was 20.0%(7/35).The concordance rate of the DPO-PCR tests between the tissue samples from the RUT kit and the gastric biopsy samples was 94.4%(51/54).The rate of DPOPCR and silver stain positivity in the RUT-negative cases was 20.0%(7/35).CONCLUSION:In diagnosing H.pylori infection,DPO-PCR can be performed on tissue samples that have been processed by the RUT kit.Particularly,in patients with RUT-negative results,DPO-PCR on these tissue samples could be helpful in detecting of H.pylori infection.

Diagnostic assays developed for the control of foot-andmouth disease in India

Foot-and-mouth disease(FMD) is a highly contagious and economically devastating disease of livestock, primarily affecting cattle, buffalo and pigs. FMD virus serotypes O, A and Asia1 are prevalent in India and systematic efforts are on to control and eventually eradicate the disease from the country. FMD epidemiology is complex due to factors like co-circulation, extinction, emergence and re-emergence of genotypes/lineages within the three serotypes, animal movement, diverse farm practices and large number of susceptible livestock in the country. Systematic vaccination, prompt diagnosis, strict biosecurity measures, and regular monitoring of vaccinal immunity and surveillance of virus circulation are indispensible features for the effective implementation of the control measures. Availability of suitable companion diagnostic tests is very important in this endeavour. In this review, the diagnostic assays developed and validated in India and their contribution in FMD control programme is presented.

Establishment of a Multiplex PCR System to Diagnose Tuberculosis and Other Bacterial Infections

In order to rapidly diagnose and differentiate tuberculosis from other bacterial infections, a 16S rRNA gene (16s rDNA)-directed multiplex PCR system was developed. In this system, a pair of universal primers and a tubercle bacillus (Tb)-specific primer were designed based on highly con- served regions and Tb species-specific variable region of bacterial 16s rDNA. A 360bp fragment was detected in all bacteria tested, and a 210bp fragment was found only in Tb. 19 species of known bac- teria including Tb were used for evaluating specificity, universality and sensitivity of the PCR. Candi- da albicans and human diploid cell served as controls. It was found that both 210bp and 360bp frag- ments were amplified only in Tb, and only 360 bp fragment was detected in other 18 species of gener- al bacteria. Candida albicans and human cells were negative for both 360bp and 2l0bp fragments. The lowest detectable level of the PCR was 10 fg of DNA for Escherichia coli and 100 fg of DNA for Tb. The results indicated that this multiplex PCR system for the simultaneous detection of Tb and other common bacteria had higher specificity and sensitivity, as well as good universality and might be useful to rapidly diagnose bacterial infections and effectively distinguish tuberculosis from other bacterial involvement.