Development of a fungus-specific PCR assay for detecting low-level fungi in an indoor environment

A fungus-specific PCR assay using only one primer set has been developed for detecting indoor fungi. Four fungal primer sets, NS3/NS4, NS5/NS6, FF1/FR1 and FF2/FR1, were tested with DNA from humans, rats, mice, bacteria, pollens and six commonly found fungal species (Alternaria chamydospora, Aspergillus flavus, Candida famata, Cladosporium fermentans, Penicillium chrycoIgenum and Stachybotrys chartarum). Results indicated that, although all four primer sets could amplify the fungal DNA, only FF2/FR1 demonstrated no cross-amplification with non-fungal DNA. In addition, these amplified fragments were sequenced to ensure that they indeed matched known fungal DNA sequences. Furthermore, besides the tested fungi, eighteen more genera of fungal sequences were examined and found to match the FF2/FR1. Here, the method of bead-beating was identified as the most effective way for spore breakage and fungal DNA release. The PCR amplification efficiency and potential inhibition were examined using different process solutions and preparation procedures. It was found that, when using 20% nutrient media and homogenization-first procedure, a higher amplification efficiency with less inhibition was achieved. Although positive bands were observed at 0.2 fungal spore/reaction using the homogenization-first procedure, the sensitivity of this assay would be two fungal spores/reaction for environmental samples.     

Identification of different bacterial DNAs in human coronary arteries

BACKGROUND: Various studies have suggested a link between infection, atherosclerosis and coronary artery disease. We studied whether bacterial DNA is present in coronary specimens obtained from left anterior descending coronary arteries of subjects having sudden deaths of cardiovascular and other causes, as verified by an autopsy. MATERIALS AND METHODS: Coronary specimens were obtained from five subjects who died of sudden coronary causes and five controls. Broad-range 16-s rDNA PCR (Br-PCR) amplification, cloning and sequencing were used to detect bacterial rDNA. RESULTS: Bacterial rDNA sequences of oral pathogens were detected from the coronary samples in all cases regardless of the cause of death. CONCLUSIONS: Br-PCR is a powerful method to detect bacterial rDNA. By this method we were able to detect wide palette of oral bacteria from coronary tissues. Our findings suggest that atheromas may act as mechanical sieves collecting bacteria from the circulation.     

Detection of human apolipoprotein E3, E2, and E4 genotypes by an allele-specific oligonucleotide-primed polymerase chain reaction assay: development and validation.

A polymerase chain reaction (PCR) assay has been developed and validated by using allele-specific oligonucleotide (ASO) primers to specifically amplify E3, E2, and E4 polymorphic sequences of the human apolipoprotein E (apo E) genes. Degenerate ASOs containing one or two additional 3' mismatches provided greater specificity than did ASOs containing a single mid-sequence or 3' allele-specific mismatch with plasmid pEB4 or genomic DNA as template. Optimal specificity and efficiency of amplification did not correlate with primer annealing conditions, whether determined theoretically or via oligo-melting experiments. Pre-cycling denaturation times and high cycling denaturation temperatures were also required for optimal amplification, presumably because of the high G:C content (75-85%) of apo E gene sequences. Conditions permissive for amplification and discrimination with plasmid DNA did not transpose favorably to amplification from human genomic DNA from peripheral blood leukocytes; the latter required nested primer reactions. These data may be valuable in predicting PCR assay conditions for other G:C-rich sequences containing polymorphic sequence differences. The assay described is both more accurate and rapid (24 h) than previously described methods for phenotyping or genotyping human apo E from blood specimens.     

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection

The development of a quadriplex PCR method with amplification of HCMV in a single‐step procedure using primers taken from four different regions of the viral genome is described. Different concentrations of dNTPs and MgCl₂ were assayed in order to optimize the constitution of the buffer for the multiplex PCR. The specificity of the PCR was tested with 100ng, 10ng, and 1ng of genomic MRC‐5 cell DNA infected with CMV in the presence of 10μg of uninfected MRC‐5 cell DNA. The sensitivity of the PCR was evaluated by the amplification of various amounts (100ng, 10ng, 1ng, and 0.1ng) of genomic MRC‐5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10ng of genomic MRC‐5 cell DNA with each primer pair. When all four sets of primers were combined in a single reaction tube, the sensitivity of the assay was equivalent to 10ng of genomic MRC‐5 cell DNA, whereas amplification from 1ng genomic MRC‐5 cell DNA produced only a subset of the amplimers. By amplifying four target‐sequences of HCMV simultaneously with minimum incubation time at each temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allowed the detection of variants and achieved maximal sensitivity and specificity which are essential for a diagnostic utilization. J. Clin. Lab. Anal. 13:99–105, 1999. © 1999 Wiley‐Liss, Inc.     

TaqMan-MGB探针实时荧光定量-聚合酶链反应检测肺炎支原体方法的建立

目的 建立敏感、特异的TaqMan-MGB探针实时荧光定量-聚合酶链反应(real-time PCR)快速检测方法,为临床标本中肺炎支原体快速检测提供技术支持。方法 选取肺炎支原体重复序列RepMP23中保守区域设计、合成特异性扩增引物和TaqMan-MGB探针,建立并优化real-time PCR检测方法。对优化后的方法使用标准浓度核酸进行扩增效率、灵敏度及特异度评价,并与已报道的肺炎支原体荧光PCR方法进行比较。结果 本研究建立的TaqMan-MGB探针荧光PCR方法对肺炎支原体的检测限为0.2 cfu,比普通TaqMan real-time PCR检测限(3～5 cfu)提高了一个数量级。其检测标准曲线的循环阈值(Ct) 与模板拷贝数呈良好的线性关系(R2=0.999)。用该方法扩增23种呼吸道常见病原菌染色体及人类染色体,结果均为阴性,特异度为100%。结论 TaqMan-MGB探针real-time PCR方法可快速、灵敏、特异地检测肺炎支原体,有很好的应用前景与价值。     

小儿常见感染菌16S—23S rRNA基因区间的分子生物学研究

目的：探讨聚合酶链反应（PCR）加限制性内切酶片段长度多态性（RFLP）分析在细菌rDNA区间检测中的应用。方法：以16S－23S RNA基因区间为靶序列,设计引物,选择合适的内切酶,采用PCR法加RFLP法检测标准菌株及临床菌株的rDNA区间。结果：26株不同的标准菌株经PCR扩增后,分别出现一条带,两条带,三条带及多条带的不同DNA图谱,敏感性试验可检出2．5CFU的细菌,与人类基因组DNA,真菌及病毒无交叉反应,其中14种菌经一步PCR扩增即可区分,另12种菌除肺炎克雷伯氏菌与坚韧肠球菌经Hinf I或Alu I酶切后仍不能区分外,其余经其中一内切酶酶切后均能区分,32例血培养阳性标本均扩增出与相应标准菌株相一致的图谱。结论：16S－22S rRNA区间基因PCR．扩增加RFLP技术检测细菌rDNA区间,具有特异,敏感,快速,准确的特点,为细菌感染的病原诊断提供新的科学依据。     

Evaluation of inhibition and cross-reaction effects on real-time PCR applied to the total DNA of wastewater samples for the quantification of bacterial antibiotic resistance genes and taxon-specific targets

In order to evaluate the applicability of six primer and probe sets for TaqMan real-time RCR on DNA of wastewater samples, effects of the sample matrix and DNA background on target quantification were studied with respect to differences between functional genes and taxonomically used rDNA targets. Primer/probe assays for real-time PCR (TaqMan) designed to quantitatively detect the antibiotic resistance genes bla(VIM), vanA, ampC, mecA, and taxon-specific 23S rDNA sequences for Pseudomonas aeruginosa and Enterococcus faecium/faecalis were tested for their sensitivity and amplification robustness. The amplification of their gene targets in DNA extracts of wastewater ("wastewater DNA") and reference strains ("reference DNA") was compared with their amplification in "model DNA" which was composed of wastewater DNA and reference DNA. Target detection was quantifiable along up to seven decimal orders of magnitude. For the detection of the resistance genes bla(VIM), vanA, ampC, and mecA as well as the enterococci directed PCR only weak or no inhibition due to the impurities or wastewater DNA matrix were demonstrated for the applied target concentrations. The taxonomically applied detection system for the quantification of P. aeruginosa showed a limited performance. For the analysis of the amplification dynamics of possibly similar nucleotide sequences of organisms related to P. aeruginosa a SYBR Green assay was employed. Competitive amplification of similar sequences was identified to be a major mechanism of reduced sensitivity. Hence, the primers were modified for an optimised detection. With the resulting reduction of cross reactions an increased sensitivity was achieved for the detection and quantification of P. aeruginosa in wastewater DNA.     

Development of conventional and real-time PCR assays for the rapid detection of group B streptococci

BACKGROUND: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. METHODS: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCycler(TM). For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. RESULTS: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only approximately 30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. CONCLUSION: These assays provide promising tools for the rapid detection and identification of GBS.     

Comparison of multiplex PCR,PCR-ELISA and fluorogenic 5' nuclease PCR assays for detection of plasmid-bearing virulent Yersinia enterocolitica in swine feces

Swine are implicated as the principal animal reservoir for plasmid-bearing Yersinia enterocolitica (YEP(+)) strains that are pathogenic to humans. To evaluate the utility of the PCR for detection of YEP(+) strains in naturally-contaminated pig feces, samples were first enriched in Irgasan ticarcillin potassium chlorate broth for 48 h at 25 degrees C and then tested by multiplex PCR, PCR-ELISA, and fluorogenic 5' nuclease PCR assays. Three different primer sets for amplification of the ail gene sequences were used in these three assays. Three out of 50 (6%) samples were positive for YEP(+) strains using the multiplex PCR targeting the chromosomal ail (170 bp) and plasmid virF (591 bp) genes. Two of the 3 samples positive by the multiplex PCR were also positive by the PCR-ELISA method using primers targeting the ail gene (425 bp). In contrast, the fluorogenic 5' nuclease PCR assay failed to detect an ail gene sequence (118 bp) in any of the 50 samples. These results indicate that the multiplex PCR was the most reliable and sensitive assay for detecting YEP(+) strains in feces among the three assays evaluated.     

半巢式聚合酶链反应-微孔板杂交法检测沙眼衣原体

目的 建立半巢式聚合酶链反应－微孔板杂交法（半巢式ＰＣＲ－ＭＰＨ）检测泌尿生殖道沙眼衣原体（Ｃｔ）,方法 分别用质粒和主要外膜蛋白基因引物进行扩增。将下游引物用生物素标记,经半巢式ＰＣＲ扩增Ｃｔ主要外膜蛋白基因后,扩增产物被包被有链霉亲和素的微孔板捕获,经碱变性后与标有荧光素的特异性探针进行杂交。然后通过辣根酶标记的抗荧光素抗体与杂交分子反应,经过酶底物显色,通过测定吸光度来判断结果。结果 主要外     

No evidence of cross-species transmission of mouse retroviruses to animal workers exposed to mice

BACKGROUND: Although recent data have brought into question the association between xenotropic murine leukemia virus‐related virus (XMRV) and chronic fatigue syndrome, one group has reported evidence of human infection with distinct polytropic murine leukemia viruses (MLVs). Occult retroviral infection among humans poses a significant public health risk should it be introduced into the blood supply. To explore the possibility of cross‐species transmission of MLVs to humans, we sought molecular and serologic evidence of XRMV/MLV infection among a cohort of animal workers highly exposed to mice. STUDY DESIGN AND METHODS: Before the commencement of the study, the laboratory and equipment were demonstrated to be free of XMRV/MLV DNA sequences. DNA extracted from 43 animal workers was tested using nested polymerase chain reaction (PCR) with published primer sets, targeting regions of XMRV and MLV gag. Negative controls were assayed in a 1:1 ratio with specimens. Serum specimens were tested using a validated immunoblot assay containing cross‐reactive XMRV antigens. RESULTS: Initial molecular assays demonstrated that the physical space and laboratory equipment were free of MLV and XMRV DNA sequences. Nested PCR assays using multiple primer sets successfully amplified XMRV and MLV sequences from positive controls with high sensitivity. A single, nonreproducible, false‐positive result from one specimen was shown to be the result of subsequent contamination. Immunoblotting of all subjects' sera failed to demonstrate any evidence of seroreactivity to XMRV proteins. CONCLUSIONS: There was no evidence of human infection with XMRV/MLV among a cohort of individuals highly exposed to mice. These data suggest that the likelihood of cross‐species transmission events of MLV from mice to humans is low.     

Evaluation of the alkaline wash/lysis procedure for the molecular diagnosis of a positive bacterial blood culture in clinical routine practice

Blood culture is commonly used to detect microorganisms in patients with a suspected blood infection. This study evaluated the alkaline wash/lysis procedure to extract DNA of microorganisms in a clinical blood culture. A multiplex polymerase chain reaction (PCR) targeting the 16S rDNA (ribosomal DNA) gene and the fungal ITS (internal transcribed spacer) gene was used as a reliable indicator for the presence of microorganism DNA in the extracts. A total of 535BacT/ALERT positive blood culture bottles were evaluated. Multiplex PCR showed positive results in 530 DNA extracts, but 5 DNA extracts gave negative results. We conclude that the alkaline wash/lysis procedure in combination with the multiplex PCR is a simple and sensitive method, which can be used in a standard diagnostic laboratory to detect microorganisms in blood culture material. J. Clin. Lab. Anal. 24:139–144, 2010. © 2010 Wiley‐Liss, Inc.     

应用巢式PCR检测实验小鼠组织中申克孢子丝菌

目的研究实验小鼠申克孢子丝菌感染的分子生物学鉴定方法,为建立检测人申克孢子丝菌感染的快速、敏感、特异方法提供依据。方法建立申克孢子丝菌感染小鼠模型,应用合成的申克孢子丝菌种特异性引物ITS3-SSP进行巢式PCR扩增小鼠皮损组织内核糖体DNA的ITS2靶序列,将检测结果与标准形态学鉴定结果对比,观察种特异性引物PCR扩增结果的准确程度、敏感性和特异性。结果组织学检查可以从11只感染小鼠尾组织中见到染成紫红色的圆形、卵圆形孢子;第一循环PCR检测实验小鼠申克孢子丝菌,只有3只小鼠呈阳性;而巢式PCR检测申克孢子丝菌特异性DNA,11只实验小鼠中9只呈阳性;而3只对照组小鼠无一呈阳性。结论本实验结果说明巢式PCR结合种特异性引物ITS3-SSP可以有效检出实验小鼠皮损中的申克孢子丝菌,具有一定的敏感性和特异性,尤其是对于组织学检查和真菌培养阴性的标本,本方法是一个很有潜力的替代方法。     

Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.

A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.     

Comparison of 9 different PCR primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 RNA extraction methods

The sensitivity and specificity of various severe acute respiratory syndrome coronavirus (SARS-CoV) PCR primer and probe sets were evaluated through the use of commercial kits and in-house amplification formats. Conventional and real-time PCR assays were performed using a heat-block thermocycler ABI 9600, the Roche LightCycler version 1.2, or the ABI 7000 Sequence Detection System. The sensitivity of all primers was between 0.0004 and 0.04 PFU with viral cell lysate and between 0.004 and 0.4 PFU in spiked stool specimen per PCR assay. The primer sets for real-time PCR assays were at one least 1 log more sensitive than the primer sets used in the conventional PCR. A panel of viruses including swine gastroenteritis virus, bovine coronavirus, avian bronchitis virus (Connecticut strain), avian bronchitis virus (Massachusetts strain), human coronaviruses 229E and OC43, parainfluenza virus (type III), human metapneumovirus, adenovirus, respiratory syncytial virus, and influenza A were tested by all assays. All real-time PCR assays used probe-based detection, and no cross-reactivity was observed. With conventional PCR, analysis was performed using agarose gel electrophoresis and multiple nonspecific bands were observed. Two commercial extraction methods, magnetic particle capture and silica-based procedure were evaluated and the results were comparable. The former was less laborious with shorter time for completion and can easily be adapted to an automated system such as the MagNa Pure-LC, which can extract nucleic acid from clinical samples and load it into the sample capillaries of the LightCycler. As exemplified by this study, the continued refinement and evaluation of PCR procedures will greatly benefit the diagnostic laboratory during an outbreak of SARS.     

Direct detection of Mycobacterium tuberculosis in clinical specimens using single-tube biotinylated nested polymerase chain reaction-enzyme linked immunoassay (PCR-ELISA)

A biotinylated single-tube nested polymerase chain reaction (PCR) assay with microwell hybridization assay (bPCR-ELISA) was developed for detection of Mycobacterium tuberculosis in clinical specimens. A total of 659 specimens (601 respiratory specimens and 58 nonrespiratory specimens) were collected for evaluation using three DNA amplification techniques: newly designed bPCR-ELISA, in-house single-tube nested PCR for IS6110 gene sequence (nPCR), and commercial automated assays, the Cobas Amplicor System from Roche Diagnostic Systems (aPCR). Sixty-four (9.7%) specimens were culture-positive for M. tuberculosis. Eleven (1.7%) specimens culture-positive for nontuberculosis mycobacteria were negative by all three PCR assays. The resolved performance of bPCR-ELISA, nPCR, and aPCR was found at sensitivities of 97%, 94%, and 97%, respectively. All three PCR assays exhibited a 100% specificity. In evaluation of bPCR-ELISA, a clear distinction between PCR-positive and PCR-negative specimens when an OD405 value of 0.6 was chosen as cut-off. With serial dilutions of M. tuberculosis H37Rv DNA, the detection limit of bPCR-ELISA was found to be 0.75 cfu per reaction at OD405 value of 0.6. Our developed bPCR-ELISA provides a highly sensitive and low-costing molecular diagnosis suitable for developing countries with high prevalence of tuberculosis.     

A highly sensitive immuno-polymerase chain reaction assay for human angiotensinogen using the identical first and second polyclonal antibodies

We describe an immuno-polymerase chain reaction (immuno-PCR) assay for the detection of human angiotensinogen using identical first and second polyclonal antibodies. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound with streptavidin to biotinylated second antibody. Human recombinant angiotensinogen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. To reduce the effect of nonspecific amplification, the optimal concentrations of streptavidin and DNA label were determined to be 0.1 mg/l and 0.5 ng/l, respectively. The detection limit of the immuno-PCR assay was 0.1 ng/l, an approximately 2.5x10(5)-fold improvement compared with a conventional enzyme-linked immunosorbent assay. These results indicate that a highly sensitive immuno-PCR for human angiotensinogen can be developed even with identical first and second polyclonal antibodies.     

Identification of predominant human and animal anaerobic intestinal bacterial species by terminal restriction fragment patterns (TRFPs): a rapid, PCR-based method

Identification of predominant human and animal intestinal tract anaerobes by conventional methods is cumbersome, time-consuming and less sensitive as compared to molecular methods. We have developed a molecular technique to identify most of the abundant intestinal microflora by polyermase chain reaction (PCR) amplification of a 16S rRNA gene fragment using a pair of universal PCR primers. The forward PCR primer was labelled with 6-carboxyfluorescein amino hexy (6-FAM) fluorescent dye to detect the terminal fragment of the PCR products after digestion with restriction enzymes. The PCR products were purified and digested with restriction enzymes and were analysed by capillary electrophoresis using an automated DNA sequencer. The data was analysed with GeneScan software 2.1. Eleven bacteria (Eubacterium biforme, E. limosum, Peptostreptococcus productus, Lactobacillus acidophilus, Bacteroides thetaiotaomicron, B. vulgatus, B. distasonis, Clostridium clostridiiforme, C. leptum, C. perfringens and Escherichia coli) that are predominant in human and animal intestinal tract were successfully identified by this rapid molecular technique. This protocol is rapid, accurate, sensitive and capable of identifying multiple organisms in a single sample.     

Polymerase chain reaction inhibition assay documenting the amotosalen-based photochemical pathogen inactivation process of platelet concentrates

BACKGROUND: The INTERCEPT Blood System (Baxter Healthcare Corp.) for platelets (PLTs) uses amotosalen-HCl (S-59) in conjunction with ultraviolet A (UVA) light to inactivate contaminating pathogens by modifying the nucleic acids of pathogens. The success of this photochemical treatment (PCT) process can be documented indirectly with a high-performance liquid chromatography assay measuring the photodegradation of amotosalen and measurement of the UVA light dose delivered by the illumination system. STUDY DESIGN AND METHODS: To develop an assay that documents the success of PCT directly on the effector molecule DNA, the effect of PCT on PLT-derived mitochondrial DNA (mtDNA) was examined. mtDNA-specific polymerase chain reaction (PCR) assays were tested with regard to their susceptibility for PCT, their reliability in terms of PCR performance, and the absence of polymorphic sites in primer hybridization loci. RESULTS: Suitable PCR amplification targets were found in the regions of 16S rDNA, cytochrome c oxidase I, and cytochrome c oxidase III of mitochondria. Amplicon sizes between 868 and 1248 bp gave consistent signals before PCT and complete inhibition of the PCR signal after PCT. Amplicons of less than 300 bp were found to be transparent to PCT. CONCLUSION: Based on PCT-mediated mtDNA modifications in PLTs, a PCR inhibition assay was established with a large amplicon documenting the success of PCT and a small amplicon serving as an internal control.     

Detection of Plasmodium falciparum and Wuchereria bancrofti infected blood samples using multiplex PCR

A rapid and sensitive multiplex PCR has been developed for the diagnosis of multiple parasitic infection in human blood. Infection is detected by a single multiplex PCR reaction containing two pairs of oligonucleotide primers whereby each primer is specific for each parasite species. These primer sets amplified 400 and 450-bp fragments for Wuchereria bancrofti and 208-bp fragment for Plasmodium falciparum. The PCR products derived from each parasite species were visualized in ethidium bromide-stained agarose gels, therefore allowing the rapid identification of any, or all, of the two human parasites, if present, in a single amplification reaction. This multiplex PCR was very sensitive with the ability to detect the presence of as little as 10 pg of parasite DNA. The primers used in this multiplex PCR also showed highly specific amplification of each respective parasite DNA without the presence of non-specific and non-target PCR products. This multiplex PCR system was used to analyse 36 human blood samples of Myanmar workers in the endemic area at Tak Province, Thailand. Two samples showed the multiple infection, 27 samples were either infected with W. bancrofti or P. falciparum and seven samples were negative for both methods. The high sensitivity, specificity and rapidity of this multiplex PCR method make it suitable for large-scale epidemiological studies and following of drug treatment.