人类血小板抗原1～6系统同步基因分型的研究

为研究采用PCR—SSP技术,建立可靠的人类血小板抗原HPA-1,2,3,4,5,6系统的同步基因分型方法,并以所建立的方法研究血小板抗原。设计合成18条序列特异性引物,探索最佳退火温度,通过调整引物浓度、Mg2＋离子浓度,使HPA-1~6系统等位基因在同一条件下进行同步扩增和扩增产物在同一凝胶中进行同步电泳。引物的特异性和灵敏度采用基因型已知的质控DNA进行验证。应用此方法,对2000年度国际输血协会（ISBT）第十届血小板基因定型与血清学工作组送检的15份考核样本（其中血样2份,DNA样本13份）进行了基因分型。用此方法检测质控DNA,结果与已知的HPA基因型完全相符;15份第十届血小板基因定型与血清学工作组的考核样本的检测结果,与ISBT公布的结果完全相同,准确率达100%。Abstract: To set up the simultaneous genotyping of human platelet antigens of 1,2,3,4,5,6 system by PCR—SSP assay and use the genotyping method for the study of platelet antigens. In this study, 18 sequence-specific primers were designed and synthesized. The annealing temperature for all sequence-specific primer pair, the concentration of each primer pair and the concentration of Mg2+ were adjusted to the optimum so that HPA-1 to 6 systems could be amplified simultaneously under the same PCR cycling parameters. The electrophoresis of PCR products was conducted simultaneously on the same agarose gel. Control DNA samples that genotypes known were used to confirm the sensitivity and specificity of each sequence-specific primer. 15 coded samples (including 2 blood samples and 13 DNA samples) distributed by 10TH Platelet Genotyping and Serology Workshop of the International Society of Blood Transfusion (ISBT) were typed for HPA-1 to 6 systems by this method. A concordance rate of 100 percent was observed between the results of control DNA samples typed by our PCR—SSP assay and the data of known specificity of control DNA. The results of 15 coded samples tested by our method agreed well with the results provided by ISBT report.     

Investigation of sensitivity,specificity and accuracy of Tetra primer ARMS PCR method in comparison with conventional ARMS PCR,based on sequencing technique outcomes in IVS-II-I genotyping of beta thalassemia patients

Purpose

Beta thalassemia is one of the most important hematic diseases all around the world and solving the problems caused by this abnormality is strongly dependent on precise detection and reliable screening of high-risk couples. The aim of our study was the investigation of sensitivity, specificity and accuracy of Tetra primer ARMS PCR method comparing with conventional ARMS PCR, based on sequencing technique outcomes for genotyping of IVS-II-I mutation in beta thalassemia patients.

Methods

Fifty seven samples including two homozygote, 49 heterozygote and 6 normal specimens were analyzed by Tetra primer ARMS PCR and conventional ARMS PCR methods. DNA was extracted by the standard method of salting out for leukocyte genomic DNA extraction of blood specimens and a high pure PCR template preparation kit was used for DNA purification of CVS samples. The results obtained by Tetra primer ARMS PCR and conventional ARMS PCR methods were compared with gold standard technique, i.e. sequencing.

Results

All three parameters including specificity, sensitivity and accuracy were 100% for Tetra primer ARMS PCR method, while they were 100%, 92.45% and 92.7% for conventional ARMS PCR technique respectively. Comparing with Tetra primer ARMS PCR which represented 100% agreement with sequencing method, conventional ARMS PCR technique only showed 47.1% agreement, because of 4 discordant results.

Conclusion

Tetra primer ARMS PCR method is an almost reliable, sensitive and accurate technique and it is suggested that it can be used as a complementary method for diagnostic cases instead of conventional ARMS PCR method. This suggestion originated with perfect rate of agreement between outcomes of sequencing method, as a gold standard method of detecting the mutations, and Tetra primer ARMS PCR technique comparing with conventional ARMS PCR method.     

垃圾填埋场中厌氧真菌18SrDNA的PCR扩增及鉴定

采用机械破壁法直接从来自7个不同地区的垃圾填埋场滤液样本中提取真菌DNA,应用真菌通用引物NS1和NS8扩增18SrDNA（约1800bp),多聚酶链式反应（PCR）产物的琼脂糖凝胶电泳结果表明所有的样本均得到了扩增;以PCR产物作为模板,采用厌氧真菌Chytridiomycetes科的专用引物Chyt-719和Chyt-1553进行二次PCR扩增（约857bp),该阳性扩增产物克隆和测序结果首次表明在食草动物瘤胃中存在的厌氧真菌Chytridiomycetes也存在于垃圾填埋场中,且为Neocallimastix属。     

NLF2 gene expression in the endometrium of patients with implantation failure after IVF treatment

We developed and evaluated the specificity and sensitivity of a loop-mediated isothermal amplification (LAMP) method for rapid detection of the multidrug-resistance gene cfr. A pair of outer primers and a pair of inner primers and one loop primer were specially designed for recognizing seven distinct sequences on the target cfr gene. The amplification reaction was performed within only 35 min under isothermal conditions at 63 °C in a regular water bath with visual measurement. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of 1 pg per tube of chicken Staphylococcus sciuri genomic DNA. The detection rate of cfr gene for 50 Staphylococcus clinical strains by LAMP assays was 16% and appeared 100% consistence with the result by PCR method. The LAMP method reported here demonstrated a potential and valuable means for detection of the multidrug-resistance gene cfr: easy, rapid, visual, specific, accurate and sensitive, especially useful for on-the-spot investigation.     

人趋化因子MIP-3α的原核可溶性表达及趋化活性分析

目的:克隆人趋化因子MIP3α,进行原核表达并初步鉴定其趋化活性。方法:从人扁桃体中提取总RNA,进行RTPCR,扩增MIP3α成熟蛋白基因,重组于pET32a(+)载体,转化大肠杆菌BL21TrxB(DE3),进行融合表达,Westernblot验证融合蛋白,金属离子亲和层析,肠激酶酶切,弱阳离子交换层析,得到纯化的MIP3α蛋白,趋化试验鉴定其趋化活性。结果:成功构建了MIP3α天然蛋白的硫氧还蛋白融合表达载体,表达并纯化出MIP3α蛋白,Westernblot证明融合蛋白能与羊抗人MIP3α抗体结合,纯化的MIP3α蛋白能趋化HEK293CCR6稳定转染细胞。结论:构建的天然MIP3α融合表达载体以可溶性蛋白的方式稳定表达MIP3α,初步纯化得到的MIP3α具有趋化HEK293CCR6稳定转染细胞的活性。     

Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR

This study was designed to develop a multiplex PCR method with five specific primer pairs for the detection of Salmonella spp., Salmonella subspecies I, Salmonella enterica serovars Typhimurium, Typhi and Enteritidis. A multiplex PCR was constructed with five primer pairs for the detection of Salmonella and pathogenic Salmonella serovars, including a specific primer pair for Salmonella Typhi, based on the sequence comparison between genomic DNA sequences of 12 Salmonella strains. Each primer pair was specifically targeted to Salmonella spp., Salmonella subspecies I, Salmonella Typhimurium, Typhi and Enteritidis. This multiplex PCR was evaluated with various DNAs of Salmonella serovars that yielded high specificity for amplifying the expected PCR products of Salmonella serovars. Using this primer pair, a set of multiplex PCR was performed for the rapid identification of salmonellae and major pathogenic Salmonella serovars. Although this multiplex PCR method will need to be evaluated for a wide range of Salmonella serovars among multilaboratories, it should be useful for identifying clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological testing.     

Rapid and specific identification of Shiga toxin-producing Escherichia coli in faeces by multiplex PCR

AIMS: The object of this study was to develop a multiplex PCR system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) in faeces. METHODS AND RESULTS: A multiplex PCR (mPCR) protocol was developed using a primer pair specific for genes that are involved in the biosynthesis of the O157 E. coli antigen, and primers that identify the sequences of Shiga toxin 1 and 2 (stx 1 and stx1) and the intimin protein (eaeA). The mPCR assay was used for amplification of STEC genes in bacteria directly (after enrichment) in faeces. The test was very sensitive and could detect between 9 and 1 bacterial cells per gram of faeces. The mPCR was used for the examination of 69 bovine faecal samples derived from healthy cattle. The results indicated that 62 x 3% of the samples were positive, generating at least one PCR amplicon of the expected size. CONCLUSIONS: The method can be applied for rapid and specific identification of STEC bacteria in faecal samples, and for differentiation of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect Shiga toxin-producing E. coli directly in faeces within a short time represents a considerable advancement over more time-consuming and less sensitive methods for identification and characterization of STEC bacteria.     

Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 microl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.     

Investigation of animal botulism outbreaks by PCR and standard methods

Abstract A double PCR procedure is proposed for identification of Clostridium botulinum C and D. This method consists of a first PCR amplification with a degenerate primer pair able to amplify a 340 bp common DNA fragment from botulinum neurotoxin (BoNT) C1 and D genes, followed by two subsequent PCR amplifications with two primer pairs specific for BoNT/C1 and D respectively (198 bp DNA fragment). This method was found to be specific for C. botulinum C and D, amongst 81 strains of C. botulinum and 21 different species of other Clostridium and bacteria tested. The detection limit ranged from 10 to 10³ bacteria in the reaction volume according to the C. botulinum C and D strains. In 160 naturally contaminated animal and food samples submitted to a 48 h enrichment culture, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay. A clear distinction between botulism type C and D was obtained. The double PCR provides a reliable alternative for detection and identification of C. botulinum C and D in clinical and food samples.     

Designing primers from multiple sequences using matchup program to improve detection of hepatitis B virus by polymerase chain reaction

Traditionally primers for PCR detection of viruses have been selected from genomic sequence of single or representative viral strain. However, high mutation rate of viral genomes often results in failure in detecting viruses in clinical and environmental samples. Thus, it seems necessary to consider primers designed from multiple viral sequences in order to improve detection of viral variants. Matchup is a program intended to select universal primers from multiple sequences. We designed using Matchup program primer pairs for HBV detection from 691 full genomic HBV DNA sequences available from NCBI GenBank database. Thousands of primer candidates were initially extracted and these were sequentially filtered down to 5 primer pairs. These primer pairs were tested by PCR using 5 HBV Korean HBsAg(+) patient sera, and eventually one universal primer pair was selected and named MUW (multiple-universal-worldwide). This primer pair, 3 HBV reference primer pairs reported by others and 1 commercial primer pair were compared using 86 HBV HBsAg(+) sera from Korean and Vietnamese patients. The detection rate for MUW primer pair was 72.1%, much greater than those obtained by reference and commercial primers (32.5 to 40.7%). The superiority of MUW primer pair appeared to be correlated with the conserved sequences of the forward primer binding sites and primer quality score. These results suggest that the universal primers designed by the Matchup program from multiple sequences could be useful in detecting viruses from clinical samples.     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Classification of a Brevundimonas strain detectable after PCR with a Helicobacter-specific primer pair

In a study for the isolation of new Helicobacter strains, biopsy samples were taken from the gastric mucosa of dogs and subjected to PCR amplification using a Helicobacter-specific primer pair (H276f and H676r, directed to the 16S rDNA) to identify members of this genus in the specimens. From one Helicobacter positive sample, a bacterial strain was isolated which displayed a characteristic band after PCR amplification with the Helicobacter-specific primer pair. The isolate designated H2/98-FUNDUS was motile, oxidase-, catalase- and aminopeptidase-positive and grew only under microaerophilic conditions at 37 degrees C. The bacterium was classified by a polyphasic approach, including analysis of the isoprenoid quinones, fatty acids, polar lipids and partial 16S rDNA sequence. Analysis of the 16S rDNA sequence (1003 bases) indicated that the strain H2/98-FUNDUS is a member of the genus Brevundimonas and most closely related to Brevundimonas aurantiaca DSM 4731T (99.5% sequence similarity). Isolate H2/98-FUNDUS contained a predominant ubiquinone Q-10 and a fatty acid profile with the major compounds C18:1 and C16:0. In the polar lipid extract, phosphatidylglycerol, six unknown phospholipids, one unknown phosphoglycolipid, two unknown glycolipids and two unknown aminolipids were detected. All these results indicate that H2/98-FUNDUS represents a new member of the genus Brevundimonas which gives a positive signal upon PCR employing the Helicobacter-specific primer pair.     

Identification of aster yellows phytoplasma in garlic and green onion by PCR-based methods

In the summer of 1999, typical yellows-type symptoms were observed on garlic and green onion plants in a number of gardens and plots around Edmonton, Alberta, Canada. DNA was extracted from leaf tissues of evidently healthy and infected plants. DNA amplifications were conducted on these samples, using two primer pairs, R16F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. DNA samples of aster yellows (AY), lime witches'-broom (LWB) and potato witches'-broom (PWB) phytoplasmas served as controls and were used to determine group relatedness. In a direct polymerase chain reaction (PCR) assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected amplified products of 1.2 kb. Dilution (1/40) of each of the latter products were used as template and nested with specific primer pair R16(1)F1/R1. An expected PCR product of 1.1 kb was obtained from each phytoplasma-infected garlic and green onion samples, LWB and AY phytoplasmas but not from PWB phytoplasma. An aliquot from each amplification product (1.2 kb) with universal primers was subjected to PCR-based restriction fragment length polymorphism (RFLP) to identify phytoplasma isolates, using four restriction endonucleases (AluI, KpnI, MseI and RsaI). DNA amplification with specific primer pair R16(1)F1/R1 and RFLP analysis indicated the presence of AY phytoplasma in the infected garlic and green onion samples. These results suggest that AY phytoplasma in garlic and green onion samples belong to the subgroup 16Sr1-A.     

A novel multiplex PCR assay for Salmonella subspecies identification

Aim:  To develop a novel multiplex polymerase chain reaction (PCR) assay with six primer pairs for Salmonella subspecies identification.
Methods and Results:  Five primer pairs were chosen to detect the genes ( fljB , mdcA , gatD , stn and STM4057) responsible for several phenotypic traits or encoding (sub) species-specific regions. A primer pair for invA was added to simultaneously detect Salmonella . The combination of these primer pairs was expected to give unique results to all subspecies, including Salmonella bongori. The multiplex PCR assay was optimized and evaluated with 53 Salmonella strains representing all S. enterica subspecies, S. bongori and five non- Salmonella strains. The multiplex PCR assay revealed that the genotypes were well correlated with the phenotypes in the Salmonella strains tested. The unique band patterns to their subspecies were generated from 94·3% (50/53) of the Salmonella strains, and no product from other strains by the multiplex PCR assay.
Conclusions:  The multiplex PCR assay we developed was found to be a rapid, specific and easy to perform method compared with traditional biochemical tests for Salmonella subspecies identification, especially for rapid screening of large numbers of samples.
Significance and Impact of the Study:  The assay will be useful for characterizing Salmonella isolates from reptiles, which belong to various subspecies, and therefore add to the scientific understanding of reptile-associated Salmonellosis.     

Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.     

Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood

Brucellosis is a widespread zoonosis. Currently the diagnosis of this zoonosis is based on microbiological and serological laboratory tests. Polymerase chain reaction (PCR) has been used to detect DNA from Brucella. Different target genes, primer pairs, PCR techniques, and extraction procedures have previously been published for Brucella detection. But only a few of these primers have been used in human samples, and only one study has been carried out to compare sensitivity between them. In the present study, 3 sets of primers and 3 different PCR protocols amplifying 3 different regions of the Brucella genome were compared for detection of Brucella DNA in a peripheral-blood PCR assay to conclude which is most suitable for the clinical diagnostic laboratory. These 3 pairs of primers amplify 3 different fragments included in (i) a gene encoding a 31 kDa Brucella abortus antigen (B4/B5), (ii) a sequence 16S rRNA of B. abortus (F4/R2), and (iii) a gene encoding an outer membrane protein (omp-2) (JPF/JPR). Some modifications on the reported techniques were applied during the present work to improve the outcome. The results showed that the B4/B5 primer pair had the highest sensitivity for detection of positive samples (98%), the JPF/JPR primer pair detected 88.4% of positive samples, whereas F4/R2 primer pair was the least sensitive, being able to detect only 53.1% of positive samples. The specificity of the 3 techniques was 100%. The B4/B5 primer pair was also able to detect the smallest number of bacteria (700 cfu/mL), whereas JPF/JPR was able to detect 7 x 105 cfu/mL and F4/R2 was able to detect 7 x 107 cfu/mL. It is thus concluded that using the B4/B5 primer PCR with the suggested modifications is a robust assay, which meets the sensitivity requirements to be used for testing of human blood samples for brucellosis in the diagnostic laboratory.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Influence of octanoic acid addition to medium on some volatile compounds and PR-toxin biosynthesis by Penicillium roqueforti

AIMS: The present study describes the implementation of real-time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry. METHODS AND RESULTS: Real-time PCR with Salmonella invA-specific primers and a standard bacteriological method was applied to detect Salmonella in tetrathionate enrichment cultures of 492 intestinal homogenates and 27 drag swabs from 47 poultry flocks. The number of positive individual samples by real-time PCR and culture method was 65 (12.5%) and 35 (6.8%), respectively. The number of Salmonella-positive flocks was 13 (27.7%) by both methods. PCR detection required 25 min for up to 32 samples. Melting curve analysis revealed the Tm for Salmonella-specific PCR product as 87 +/- 1 degrees C. CONCLUSIONS: Implementation of real-time PCR to tetrathionate broth enrichment step reduces the Salmonella detection time to 18 h and 25 min. Isolation of Salmonella should be carried out with PCR to determine the serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR is a powerful tool in rapid and accurate Salmonella monitoring in poultry companies, together with standard bacteriology.     

Rapid detection of human fecal Eubacterium species and related genera by nested PCR method

PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species-specific primers, would allow rapid detection of Eubacterium species in human feces.