Identification of bovine Neospora parasites by PCR amplification and specific small-subunit rRNA sequence probe hybridization.

Neospora is a newly recognized genus of pathogenic coccidia, closely related to Toxoplasma gondii, that can cause abortion or congenital disease in a variety of domestic animal hosts. On the basis of the small-subunit rRNA gene sequences of Neospora spp. and other apicomplexa coccidia, oligonucleotide primers COC-1 and COC-2 were used for PCR amplification of conserved sequences of approximately 300 bp in size. A Neospora-specific chemiluminescent probe hybridized to Southern blots of amplification products from Neospora DNA but not to Southern blots with amplified DNA from the other coccidian parasites tested. A Toxoplasma-specific probe whose sequence differed from that of the probe for Neospora spp. by a single base pair was used to distinguish these parasites by specific Southern blot hybridization. The PCR system detected as few as one Neospora tachyzoite in the culture medium or five tachyzoites in samples of whole blood or amniotic fluid spiked with Neospora parasites. In addition, Neospora PCR products were successfully amplified from whole blood and amniotic fluid samples of experimentally infected bovine and rhesus macaque fetuses. These results indicate that this PCR and probe hybridization system could be a valuable adjunct to serology and immunohistochemistry for the diagnosis of Neospora infections in bovine or primate fetuses.     

Application of Real-Time Fluorescent PCR for Quantitative Assessment of Neospora caninum Infections in Organotypic Slice Cultures of Rat Central Nervous System Tissue

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection.     

Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR

We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10(-1) tachyzoite per assay was observed with excellent linearity (R(2) = 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques (kappa = 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection.     

Detection by PCR of Neospora caninum in fetal tissues from spontaneous bovine abortions

The routine diagnosis of Neospora caninum abortion is based upon histopathologic changes in fetal tissues and identification of tissue parasites by immunohistochemistry. Confirmation of N. caninum infection by immunohistochemistry has low sensitivity. In the present study, we examined the utility of PCR in detecting N. caninum infection in fetal tissues from spontaneous bovine abortion. DNA was obtained from fresh and formalin-fixed tissues from 61 bovine fetuses submitted for abortion diagnosis. Histopathology and immunohistochemistry determined the true status of N. caninum infection in each fetus. In formalin-fixed paraffin-embedded tissues, PCR detected N. caninum DNA in 13 of 13 true-positive fetuses (100%) and in 1 of 16 true-negative fetuses (6%). In fresh or frozen tissues, PCR detected N. caninum DNA in 10 of 13 true-positive fetuses (77%) and 0 of 11 true-negative fetuses (0%). PCR also detected N. caninum DNA in 6 of 8 fetuses that had typical lesions of N. caninum but were immunohistochemistry negative, indicating a higher sensitivity of PCR in comparison to that of immunohistochemistry. N. caninum DNA was amplified most consistently from brain tissue. PCR detection of N. caninum DNA in formalin-fixed, paraffin-embedded tissues was superior to that in fresh tissues, presumably because of the increased accuracy of sample selection inherent in histologic specimens.     

Diagnosis of Neospora caninum and Toxoplasma gondii infection by PCR and DNA hybridization immunoassay.

A recently described PCR test for the identification of Neospora caninum and Toxoplasma gondii has been further developed and optimized in view of its practicability for routine diagnostic application. The N. caninum-specific PCR was adapted to the diagnostic operating standard of the T. gondii-specific PCR in that the uracil DNA glycosidase system was introduced, which eliminates potential carry-over contaminations of amplified target DNA from previous reactions. Furthermore, both PCR tests were optimized by including a DNA hybridization immunoassay based on the use of the commercially available Gen-eti-k DEIA kit. This assay allowed highly sensitive and specific detection of respective DNA amplification products and thus substantially facilitated the reading and interpretation of the test results.     

Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus

A polymerase chain reaction (PCR) amplification method was developed and evaluated to detect porcine parvovirus (PPV). A pair of 20-base primers and an oligonucleotide probe were derived from the DNA sequences common to two isolates of PPV, NADL-8 and NADL-2. The primers flanked 118-bp nucleotides within the region coding for the major structural protein VP2. After DNA amplification of PPV replicative form (RF), a 158-bp fragment was detected in agarose gels. This amplified fragment was shown to be specific for PPV DNA after Southern transfer and hybridization to a 20-base internal probe. The amplified fragment also contained a single EcoRI cleavage site. Various conditions, such as number of cycles and annealing temperature, were examined to optimize the conditions for detecting viral DNAs from infected cell cultures and swine fetal tissues. Four different isolates of PPV, NADL-8, NADL-2, KBSH and Kresse, and two other viruses, canine parvovirus (CPV) and pseudorabies virus (PRV), were included to determine specificity of amplification. Slot blot hybridization with a radiolabeled probe was used to evaluate the sensitivity of PCR amplification. The optimized protocol was specific for PPV detecting equally all four strains of PPV, but failing to amplify CPV or PRV sequences. The PCR method could detect at least 100 fg of viral replicative form (RF) DNA or the equivalent of 1 PFU of infectious virus. The applications of this method include routine detection of PPV in clinical samples and as a contaminant in mammalian cell lines.     

Detection of HPV-16 in cell lines and cervical lavage specimens by a polymerase chain reaction-enzyme immunoassay assay.

A gene amplification method that combines the polymerase chain reaction with detection of amplified DNA in a solution hybridization/enzyme immunoassay (PCR-EIA) was developed for HPV-16 DNA. Samples were amplified with primers for the E7-E1 region of HPV-16. Amplified DNA products were identified and quantitated by hybridization in solution with a biotinylated RNA probe. Labeled DNA/RNA hybrids were measured semiquantitatively in an enzyme immunoassay using solid phase anti-biotin antibody and liquid phase B-d-galactosidase labeled monoclonal antibody against DNA-RNA hybrids. Enzyme bound to the solid phase was quantitated with a fluorogenic substrate. The assay was linear over 2 log10 dilutions of SiHa cells and the detection limit was three copies of HPV-16 genome. The sensitivity of PCR-EIA for detection of PCR amplified products compared favorably with slot and Southern blots using a 32P-labeled RNA probe. The assay was used to assess HPV-16 infection of uterine cervix in women attending a clinic for sexually transmitted diseases. Twenty-one of the 81 specimens (25.9%), obtained by cervicovaginal lavage, were positive for HPV-16 by PCR-EIA. The assay provides a convenient means to objectively measure HPV DNA amplified with PCR.     

Tissue distribution of bovid herpesvirus-4 in inoculated rabbits and its detection by DNA hybridization and polymerase chain reaction

Summary A DNA hybridization technique, using the polyrepetitiveEcoRI L-fragment of bovid herpesvirus (BHV-4) as a probe, was developed to determine virus distribution in the tissues of BHV-4-infected pregnant rabbits. The cloned fragment did not react with the DNA of rabbits or of other herpesviruses, e.g., infectious bovine rhinotracheitis, bovine herpes virus mammillitis, and pseudorabies viruses. The detection limit was 10^–13g of DNA or approximately 600 genome equivalents of viral DNA, which indicates a level of sensitivity of one viral genome per 500 cells in our assay. Using conventional cell culture techniques, the virus was isolated from only one of fifteen infected rabbits and a few aborted fetuses. However, when organ culture or dot blot hybridization was used, BHV-4 was detected in all rabbits and their fetuses. Viral DNA was detected by DNA hybridization in spleen, ovary, uterus, lung, liver, salivary gland, lymph node, and placentome of adult rabbits and in a composite of fetal tissues. When polymerase chain reaction (PCR) was used, the virus was detected in several organs (including the nervous tissues) that were found negative by other techniques. These results indicate that blot hybridization and PCR are more sensitive than conventional techniques for studying the pathogenesis of BHV-4 in animals. The data obtained by these methods suggest that BHV-4 may be maintained in infected rabbits in a latent state in a variety of tissues including the nervous system.     

Diagnostic application of polymerase chain reaction for detection of Ehrlichia risticii in equine monocytic ehrlichiosis (Potomac horse fever).

Genomic amplification by the polymerase chain reaction (PCR) was used to identify a unique genomic sequence of Ehrlichia risticii directly in DNA isolated from peripheral-blood buffy coat cells of E. risticii-infected horses (Potomac horse fever) and from infected cell cultures. A specific primer pair, selected from a cloned, species-specific, 1-kb DNA fragment of the E. risticii genome as a template, was used for the amplification of the target DNA of 247 bp. The optimal number of 40 PCR cycles, determined by analyzing an amplification profile obtained with a constant Taq polymerase concentration, was used to achieve maximum amplification of the E. risticii DNA segment. Efficient amplification of target DNA was achieved with specimens processed by either the phenol extraction or rapid lysis method. The specificity of the amplified DNA product was confirmed by the proper size (247 bp) and appropriate restriction enzyme cleavage pattern of the amplified target DNA, as well as by the specific hybridization signal obtained by using a PCR-amplified 185-bp internal DNA probe. A 10(5)- to 10(6)-fold amplification of target DNA, which allowed detection of E. risticii from as few as two to three infected cells in culture and from a very small volume of buffy coat cells from infected horses, was achieved. This PCR amplification procedure was found to be highly specific and sensitive for the detection of E. risticii for the study of Potomac horse fever.     

A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep.

The DNA probe pCS20, which was cloned from the DNA of the Crystal Springs heartwater strain from Zimbabwe, cross-reacted with DNAs of heartwater strains from all endemic areas, including four heartwater strains from Zimbabwe, two strains from South Africa, one strain from Nigeria, and the Gardel strain from the Caribbean island of Guadeloupe. By nucleic acid hybridization, the pCS20 DNA probe detected Cowdria ruminantium DNA in all DNA preparations made from plasma samples from infected sheep before and during the febrile reaction. Synthetic oligonucleotides were prepared for amplification of specific C. ruminantium DNA sequences by the polymerase chain reaction (PCR). Amplification of two DNA products (181 and 279 bp) from pCS20 DNA and C. ruminantium genomic DNA of heartwater strains was demonstrated. In contrast, amplification of these products or any other products was not possible from genomic DNAs of Anaplasma marginale, Babesia bigemina, Trypanosoma brucei brucei, Escherichia coli, and bovine endothelial cells. The cross-reactivities of the 32P-labeled PCR products with genomic DNAs from several heartwater strains were similar to those with the pCS20 DNA probe. A nucleic acid-based test that uses hybridization assays and PCR provides a sensitive method for the detection of heartwater in both animals and ticks and has applications in epidemiological studies for the disease, which may allow for improved disease control.     

PCR-DNA probe assays for identification and detection of Prevotella intermedia sensu stricto and Prevotella nigrescens.

The purpose of this study was to construct PCR-DNA probe assays specific for Prevotella intermedia sensu stricto and Prevotella nigrescens based on the ability of randomly amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific markers. The strategy included four steps: (i) construction of first-generation DNA probes from a 850-bp RAPD marker for P. intermedia sensu stricto and a 1,300-bp RAPD marker for P. nigrescens, (ii) cloning and sequencing of each RAPD marker, (iii) designing of primer pairs flanking specific internal sequences of 754 bp for P. intermedia sensu stricto and of ca. 1,100 bp for P. nigrescens, and (iv) synthesis (by PCR amplification) and digoxigenin labeling of quantities of DNA probes 754 and ca. 1,100 bp in size. The PCR-DNA probe assays combine either PCR amplification of a 754-bp specific sequence in the genomic DNA of strains of P. intermedia sensu stricto and hybridization with the 754-bp digoxigenin-labeled probe or amplification of a ca. 1,100-bp sequence of P. nigrescens and hybridization with the ca. 1,100-bp probe. Specific hybridization was observed with the amplified DNAs from 25 strains of P. intermedia and 24 strains of P. nigrescens, and no reaction was observed with the PCR products from 20 foreign species. The PCR-DNA probe assays described here should allow a highly specific and sensitive detection of P. intermedia sensu stricto and P. nigrescens in mixed infections.     

Detection of bovine retrospumavirus by the polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed for detection of bovine retrospumavirus (bovine syncytial virus; BSV) provirus DNA. Two different sets of oligonucleotide primers complementary to sequences located in the gag and the pol/env gene regions were used and compared for their ability to amplify the targeted BSV sequences by PCR. The results obtained from this study have shown that it is possible to amplify the BSV provirus DNA sequences not only from total DNA of BSV-infected cell cultures, but also from total DNA of various tissues and peripheral blood mononuclear cells (PBMCs) that were collected from two rabbits experimentally infected with BSV. Sensitivities of the PCR for amplification of BSV gag and pol/env nucleic acid sequences from cell culture total DNA were 10 ng and 10 pg of DNA, respectively, as determined by the analysis of the amplified PCR products on ethidium bromide-stained agarose gels. The specificity of the PCR for both primer sets tested was confirmed when the amplified cDNA products of the expected size reacted positively with the corresponding virus-specific digoxigenin-labeled cDNA probes in Southern blot chemiluminescent hybridization assays. No amplification was obtained when the BSV-specific primers were used in the PCR with DNA material specific to either bovine leukemia virus (BLV) or bovine immunodeficiency virus (BIV) provirus genomic DNA. No cross-hybridization was obtained when the BSV-specific cDNA probes were allowed to react with BLV or BIV provirus DNA. The PCR targeting the gag and pol/env gene regions of the BSV provirus genome may be an alternative to conventional methods for the confirmation of the presence of BSV in cell cultures used for virus isolation, and for the diagnosis of BSV infection from bovine peripheral blood leukocytes.     

Detection of Pseudomonas pseudomallei by PCR and hybridization.

A molecular method for the detection of Pseudomonas pseudomallei was developed on the basis of the differences in the 23S rRNA sequences of related species of the genus Pseudomonas. An 18-base oligonucleotide probe, designed following partial sequencing of 23s ribosomal DNA (rDNA), was used for the identification and detection of P. pseudomallei either by hybridization or by direct PCR. Optimal detection was obtained by hybridization of the probe with PCR-amplified rDNA rather than with total genomic DNA or colony blots. One nanogram of template DNA amplified in a PCR mixture containing 14% glycerol could be detected in slot blots hybridized with the digoxigenin-labelled probe and the lumigen PPD detection system. Amplified rDNA sequences from 41 P. pseudomallei strains of various origins hybridized with the probe. The probe also hybridized with three Pseudomonas mallei reference strains under conditions of high stringency but failed to hybridize with amplified rDNA sequences from other closely related Pseudomonas spp. PCR with a conserved primer and the 18-base oligonucleotide probe (direct PCR) specifically amplified P. pseudomallei and P. mallei. By using these methods, approximately 10(4) P. pseudomallei cells per ml could be detected in artificially inoculated blood samples and in blood dried on filter paper following Chelex extraction. The detection limit in blood was increased to 10(2) cells per ml by concentration of bacteria from 0.5 ml of blood or by a 24-h blood culture enrichment prior to PCR. Approximately 10(3) cells per ml were detected in seeded sputum samples. The detection times by direct PCR and indirect PCR and then probe hybridization were approximately 5 h and 24 h, respectively. These results indicate that amplification of conserved rDNA sequences by PCR directly or by hybridization with a probe to PCR fragments offers promise for the detection of P. pseudomallei and P. mallei.     

Detection of JC virus by polymerase chain reaction and colorimetric DNA hybridization assay

A procedure for rapid detection of JC virus (JCV) using the polymerase chain reaction is described. The procedure was tested in eight HIV-1-seropositive patients with progressive multifocal leukoencephalopathy. One-step DNA extraction using a chelating resin was carried out on clinical samples of cerebrospinal fluid (CSF), urine and brain tissue. After amplification, PCR products were detected by a DNA hybridization method. Microplates were coated with a specific probe and hybridized PCR products were revealed by a commercial colorimetric immunoassay. Using this procedure JC virus DNA was detected in all CSF specimens from patients with progressive multifocal leukoencephalopathy. This sensitive and rapid (24 h) procedure could greatly facilitate use of the DNA probe assay for detection of JC virus in clinical laboratories.     

Diversity of host species and strains of Pneumocystis carinii is based on rRNA sequences.

We have amplified by PCR Pneumocystis carinii cytoplasmic small-subunit rRNA (variously referred to as 16S-like or 18S-like rRNA) genes from DNA extracted from bronchoalveolar lavage and induced sputum specimens from patients positive for P. carinii and from infected ferret lung tissue. The amplification products were cloned into pUC18, and individual clones were sequenced. Comparison of the determined sequences with each other and with published rat and partial human P.carinii small-subunit rRNA gene sequences reveals that, although all P. carinii small-subunit rRNAs are closely related (approximately 96% identity), small-subunit rRNA genes isolated from different host species (human, rat, and ferret) exhibit distinctive patterns of sequence variation. Two types of sequences were isolated from the infected ferret lung tissue, one as a predominant species and the other as a minor species. There was 96% identity between the two types. In situ hybridization of the infected ferret lung tissue with oligonucleotide probes specific for each type revealed that there were two distinct strains of P. carinii present in the ferret lung tissue. Unlike the ferret P. carinii isolates, the small-subunit rRNA gene sequences from different human P. carinii isolates have greater than 99% identity and are distinct from all rat and ferret sequences so far inspected or reported in the literature. Southern blot hybridization analysis of PCR amplification products from several additional bronchoalveolar lavage or induced sputum specimens from P. carinii-infected patients, using a 32P-labeled oligonucleotide probe specific for human P. carinii, also suggests that all of the human P. carinii isolates are identical. These findings indicate that human P. carinii isolates may represent a distinct species of P. carinii distinguishable from rat and ferret P. carinii on the basis of characterization of small-subunit rRNA gene sequences.     

实验感染大、小鼠和临床疑似病例肺孢子虫mt LSU rRNA基因检测

为探讨PCR技术对实验感染大、小鼠肺孢子虫肺炎(PCP)和临床疑似病例诊断的可行性。用皮下注射地塞米松法诱导SD大鼠和ICR小鼠PCP并收集临床疑似病例24h深部痰液。受试动物解剖后,制备肺印片,经瑞姬氏复合染色镜检确定肺孢子虫(Pc)感染的阳性率;同时,以针对虫体mtLSUrRNA基因设计的引物,PCR扩增鼠肺组织和支气管灌洗液(BALF),以及痰液样本中的DNA;比较实验动物样本镜检和PCR两种方法的检测结果;测定PCR的敏感性和特异性。结果表明,SD大鼠和ICR小鼠肺印片的阳性率分别为68.8%(1116)和85.7%(1821),肺组织PCR检测的阳性率分别为75%(1216)和80.9%(1721),肺泡灌洗液PCR的阳性率分别为81.2%(1316)和23.8%(521)。肺印片镜检和PCR技术检测结果无显著性差别;可见,PCR对虫体mtLSUrRNA基因的检测具有显著的特异性和敏感性,至少可以检测出0.6pg的肺孢子虫DNA;6例临床疑似病例样本中2例显示肺孢子虫PCR阳性反应。     

线粒体多态性检测寡核苷酸芯片的构建

目的构建用于线粒体DNA（mtDNA）D-环控制区（D—LOOP区）多态性多个位点集成检测的寡核苷酸芯片。方法根据GenBank中人mtDNA D—LOOP区序列设计2组引物[普通引物和N,N＇-对羧苄基吲哚三菁（Cy5）标记引物]和55种寡核苷酸探针。将探针固定于醛基修饰载玻片表面,制备mtDNA多态性检测芯片。提取40例健康人外周血标本mtDNA,应用Cy5标记引物不对称PCR扩增mtDNA D—LOOP区片段。取未变性和变性后扩增产物分别与芯片进行杂交,观察二者杂交信号强度差异,并将芯片检测结果与DNA测序结果进行比较。观察等倍稀释的不对称PCR扩增产物的杂交信号强度,检测芯片的灵敏度;将DNA测序结果与探针进行BLAST2比对,找出与PCR扩增产物完全匹配和仅1个碱基不匹配的探针,分析二者在芯片上相应位点杂交信号强度的差异,观察芯片的特异性;以放置6个月后的芯片检测外周血mtDNA,观察杂交信号强度的变化。结果不对称PCR扩增获得的mtDNA D—LOOP区片段（466bp）与预期表达片段大小一致。不对称PCR扩增产物直接杂交和变性后杂交的信号强度无明显差异,40例健康人外周血标本mtDNA的芯片杂交检测结果均与DNA测序结果完全吻合。灵敏度检测结果显示,杂交信号强度随扩增产物稀释程度的增加而减弱,芯片检测靶分子的灵敏度为0．1ng;与DNA测序结果完全匹配的探针174和仅相差1个碱基“C”的探针174c的杂交信号强度具有明显差异;而保存6个月后的芯片杂交信号强度基本保持不变。结论构建的寡核苷酸芯片可满足对mtDNA D—LOOP区多态性进行多个位点快速通量检测的需要,具有较高的灵敏度和特异性,适用于法医鉴定及线粒体疾病的检测。     

Polymerase chain reaction and DNA probe hybridization to assess the efficacy of diminazene treatment in Trypanosoma brucei -infected cattle

Four of eight Ankole longhorn cattle experimentally infected with Trypanosoma brucei were treated with 7 mg/kg diminazene aceturate (Berenil, Hoechst AG, Germany) at day 71 postinfection. The trypanocidal activity was monitored using polymerase chain reaction (PCR) and DNA probe hybridization. When extracted parasite DNA (without host DNA) was used, as little as 1 fg per reaction, which is equivalent to about 1–10% of the DNA in a single trypanosome, produced a specific product that was visible as a 177-bp band in an agarose gel. In infected cattle, specific PCR products could be amplified at as early as 1 day postinfection. PCR signals remained positive during infection, except in one sample, although aparasitemic phases occurred. In cases where treatment resulted in a significant clinical improvement, PCR signals disappeared at 3–4 days after the administration of the drug. By contrast, in cattle that showed clinical signs of CNS involvement after treatment, although aparasitemic, and died before the termination of the experiment, specific products could be amplified on several occasions following treatment. The PCR signals generated after treatment could be further enhanced by subsequent slot-blot hybridization with a T. brucei-specific DNA probe. We conclude that PCR coupled with DNA probe hybridization provides a highly sensitive tool for the assessment of therapeutic efficiency and disease progression in trypanosome infections, especially in chronic infections when the level of parasitemia is low or when trypanosomes are sequestered at cryptic sites. Received: 29 May 1998 / Accepted: 18 August 1998     

Detection and identification of mycobacteria by amplification of a segment of the gene coding for the 32-kilodalton protein.

A polymerase chain reaction (PCR) assay for the rapid detection of mycobacterial DNA is described. Oligonucleotide primers, derived from the sequence of a gene coding for the 32-kDa antigen of Mycobacterium tuberculosis, amplified DNA from all 28 species of mycobacteria tested. All nonmycobacterial species tested were negative. An oligonucleotide probe hybridized to the PCR products of the strains belonging to the M. tuberculosis complex. This method could detect as little as 50 fg, as tested with purified M. tuberculosis DNA. By this amplification method, 127 sputum specimens were tested, with 7.9% of the specimens proving to be inhibitory in PCR. The sensitivity of detection by PCR compared with that by culture was 55.9%; when the inhibitory specimens were excluded, the sensitivity was 70.4%. The specificity of PCR combined with hybridization was 100%.