Showing papers on "Multiplex polymerase chain reaction published in 1997"

Multiplex PCR: critical parameters and step-by-step protocol.

Abstract: By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions

Abstract: The present invention relates to the detection of nucleic acid sequence differences using coupled ligase detection reaction and polymerase chain reaction. One aspect of the present invention involves use of a ligase detection reaction coupled to a polymerase chain reaction. Another aspect of the present invention relates to the use of a primary polymerase chain reaction coupled to a secondary polymerase chain reaction coupled to a ligase detection reaction. A third aspect of the present invention involves a primary polymerase chain reaction coupled to a secondary polymerase chain reaction. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence differences.

Minisequencing: A Specific Tool for DNA Analysis and Diagnostics on Oligonucleotide Arrays

Abstract: We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.

Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains.

Abstract: PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.

Extended Screening by PCR for Seven cry-Group Genes from Field-Collected Strains of Bacillus thuringiensis

Abstract: An extended multiplex PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial stages of large-scale screening of cry genes by pinpointing isolates that contain specific genes or unique combinations of interest with potential insecticidal activities, thus facilitating subsequent toxicity assays. Five pairs of universal primers were designed to probe the highly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 genes. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty-two distinct cry-type profiles were identified from 126 field-collected B. thuringiensis strains. Several of them were found to be different from all published profiles. Some of the field-collected strains, but none of the 16 standard strains, were positive for cry2Ac. Three standard and 38 field-collected strains were positive by universal primers but negative by specific primers for all five known genes of cry7 and cry8. These field-collected strains seem to contain a new gene or genes that seem promising for biological control of insects and management of resistance.

Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens

Abstract: Objective To develop a multiplex polymerase chain reaction (PCR) assay to detect the genes for the major toxins of Clostridium perfringens (cpa [alpha toxin], cpb [beta toxin], etx [epsilon toxin], iA [iota toxin], and cpe [enterotoxin]). Sample population Cultures of C perfringens obtained from collections and diagnosticians throughout North America. Procedure PCR primers were derived from published sequences of the genes for the major toxins (the "typing" toxins and enterotoxin). The concentration of each primer was titrated in a PCR assay to allow concurrent amplification of multiple target sequences, and other parameters of the assay were optimized (including concentrations of other reagents and times and temperatures for denaturation of template, annealing of primers, and primer extension). Specificity of the assay was measured by comparing genotype with phenotype (where it was known). Results The genotype, determined by multiplex PCR assay, agreed with phenotype in 99% (86/87) of strains where phenotype had been determined. Applied to 361 isolates from domestic animals and human beings, 95% (n = 344) were type A, and 12.8% (n = 44) of these contained cpe. The remaining 5% (n = 17) of the isolates were type B (n = 1), type C (n = 11), type D (n = 2), or type E (n = 4). Conclusion and clinical relevance Previous studies have documented usefulness of PCR in genotyping C perfringens. The multiplex assay is as effective, but simpler, and may be a useful alternative to standard in vivo typing methods. Results of genotyping of field isolates suggested the need for further epidemiologic study of clostridial enteritis, particularly as this pertains to predominant etiologic toxin types, and documented the presence of the reportedly rare genotypes B and E.

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

Abstract: A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.

Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions.

Abstract: A multiplex PCR procedure was developed for the simultaneous detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in middle ear effusions (MEEs) from patients with chronic otitis media with effusion. The bacterial 16S rRNA gene was chosen as the target, and the procedure used one common lower primer and four species-specific upper primers. The reaction was optimized by changing the primer concentrations to yield equal amounts of amplification products. The specificity of the reaction was verified with various bacterial species found in the nasopharynx. The performance of the procedure was examined with 25 MEE specimens, and the results were compared to those obtained by conventional culture methods. A detection level of 10 bacterial cells/reaction for each of the study organisms was achieved. By conventional culture methods, 8 (32%) of the specimens showed growth of one of the study organisms. In contrast, 21 (84%) of the specimens tested positive by the multiplex PCR. None of the culture-positive specimens were PCR negative, whereas three (12%) of the PCR-positive specimens tested positive for two of the four study organisms. Thus, the multiplex PCR method improves the detection rate significantly compared to that of the conventional culture method.

Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water.

Abstract: Eight pairs of published PCR primers were evaluated for the specific detection of Cryptosporidium parvum and Giardia lamblia in water. Detection sensitivities ranged from 1 to 10 oocysts or cysts for purified preparations and 5 to 50 oocysts or cysts for seeded environmental water samples. Maximum sensitivity was achieved with two successive rounds of amplification and hybridization, with oligonucleotide probes detected by chemiluminescence. Primer annealing temperatures and MgCl2 concentrations were optimized, and the specificities of the primer pairs were determined with closely related species. Some of the primers were species specific, while others were only genus specific. Multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was demonstrated with primers amplifying 256- and 163-bp products from the 18S rRNA gene of Cryptosporidium and the heat shock protein gene of Giardia, respectively. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.

Application of Multiplex PCR and Flourescence-Based, Semi-Automated Allele Sizing Technology for Genotyping Plant Genetic Resources

Abstract: For future progress, effective conservation of plant genetic resources will require the integration of technologies and protocols that provide for the acquisition of large quantities of genetic information for improved gene and genotype identification. As a contribution to this integration, simple sequence repeat (SSR) marker analysis in Brassica spp. was evaluated as a prototype system for establishing the potential of multiplex polymerase chain reactions (PCRs) coupled with fluorescence-based DNA detection and semi-automated sizing technology for mass genotyping of plant genetic resources. Consistent results were achieved for multiplex PCRs (simultaneous amplification of several genetic markers in a single reaction) with 11 fluorescently labeled primer pairs. Comparison of computer-derived estimates of fragment sizes between gels demonstrated that automated sizing was accurate enough to achieve reliable and repeatable plant genotypes. To establish a genetic basis for putative SSR loci previously identified in Brassica spp., segregation data were analyzed. Three polymorphic SSRs in Brassica napus L. (canola and rapeseed) exhibited simple Mendelian inheritance and were not linked. One of these polymorphic loci was duplicated, i.e., a single copy marked each of the aa and cc genomes originating from progenitor species B. rapa and B. oleracea, respectively. Technologies that provide for high genetic resolution and throughput at reasonable costs will find ready application in plant genetic resources conservation. In addition, these same tools and marker systems can be applied to questions of pedigree analysis and intellectual property rights.

Specific information concerning taxonomy, pathogenicity and methicillin resistance of staphylococci obtained by a multiplex PCR.

Abstract: The use of DNA amplification techniques such as the polymerase chain reaction (PCR) in modern diagnostic microbiology not only allows the sensitive and specific identification of micro-organisms but also the detection of specific antibiotic resistance genes. This study describes a multiplex PCR on bacterial colonies picked directly from agar plates without preceding DNA preparation. Eubacteria and staphylococci were identified by 16S rRNA specific PCR products. In parallel, specific primers were used for the detection of staphylococcal coa and mecA genes. This 4-h multiplex PCR, consisting of four sets of primers, was evaluated for rapid and specific differential diagnosis of methicillin-resistant and methicillin-susceptible strains of Staphylococcus aureus and coagulase-negative staphylococci. To analyse specificity of the amplification products, 100 non-staphylococcal, eubacterial isolates and 20 Candida albicans strains were tested. In a first step, specificity of all four single sets of primers was evaluated before the co-amplification within the multiplex PCR procedure was performed. The results were compared with those of conventional susceptibility and typing methods. The specific 16S rRNA PCR product for eubacterial isolates (n = 786) and staphylococci (686) was found in all strains tested. The coa gene was detected only in S. aureus (488) strains with a specificity of 100%, and was not detected in any of the coagulase-negative staphylococci (198). The mecA gene was detected in 98% of methicillin-resistant staphylococci (393) and in 2% of all methicillin-susceptible staphylococci (293). The multiplex PCR with co-amplification of different determinants provides rapid reliable information on staphylococcal identification and methicillin susceptibility supporting the diagnosis, treatment and control of staphylococcal infections.

Application of a Nested, Multiplex PCR to Psittacosis Outbreaks

Abstract: We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.

A practical approach to genetic screening for influenza virus variants.

Abstract: This report describes a quick genetic approach to the screening of influenza virus variants. Multiplex reverse transcription (MRT) and multiplex PCR (MPCR) were used to amplify and differentiate the hemagglutinin (HA) genes of different types and subtypes of influenza viruses. Heteroduplex mobility assay (HMA) was then used to differentiate strains within the same type and subtype. Three primers complementary to the consensus 3' termini of viral genomic RNA segments of human influenza virus types A, B, and C were used in a single MRT reaction to prime the synthesis of cDNA of all the viral genome segments. The cDNA was then amplified in an MPCR containing primers for the HA genes of the H1 and H3 subtypes of type A, the HA gene of type B, and the counterpart of type C virus. Amplicons of the different types and subtypes differ in size, thus allowing typing and subtyping. The regions amplified cover most of the HA1 portion of the HA genes and therefore amplicons of variants identified by the described HMA can be sequenced directly. In the HMA, the amplicon of an individual strain was mixed with that of a reference strain and heteroduplexes derived from mismatches migrated more slowly than homoduplexes of the same size in electrophoresis, with the mobility shift pattern indicating the divergence of amplicons. The whole process from viral RNA extraction to HMA can be completed within 2 days and thus provides a quick screening before further analysis by hemagglutination inhibition testing and sequencing. In addition, all segments of the viral genome can be amplified from a single MRT reaction, which can yield valuable sources of products for future genetic analyses. This method should facilitate genetic screening and characterization of influenza virus variants.

Multiplex PCR: Critical Parameters and Step-by-Step Protocol

Abstract: By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

A multiplex PCR for identifying Shiga-like toxin-producing Escherichia coli O157:H7.

Abstract: A multiplex PCR assay specifically detecting Escherichia coli O157 : H7 was developed by employing primers amplifying a DNA sequence upstream of E. coli O157 : H7 eaeA gene and genes encoding Shiga-like toxins (SLT) I and II. Analysis of 151 bacterial strains revealed that all E. coli O157 : H7 strains were identified simultaneously with the SLT types and could be distinguished from E. coli O55 : H7 and E. coli 055 : NM, and other non-0157 SLT-producing E. coli strains. Primer design, reaction composition (in particular, primer quantity and ratios), and amplification profile were most important in development of this multiplex PCR. This assay can serve not only as a confirmation test but also potentially can be applied to detect the pathogen in food.

Direct detection of multiple point mutations in mitochondrial DNA

Abstract: Mitochondrial defects can be caused by mutations in nuclear or mitochondrial DNA. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Comprehensive molecular diagnosis requires the analysis of multiple point mutations. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDNA. The system involved three pairs of primers to amplify mutation "hot spots" at tRNA(leu(UUR)), tRNA(lys)/ATPase, and ND4 regions, followed by detection of point mutations with ASO probes. Over 2000 specimens were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. Our data demonstrate that the multiplex PCR/ASO method is much more sensitive in the detection of low mutant heteroplasmy. It is simple and cost effective, especially if a large number of samples are to be screened for multiple point mutations.

Identification of Methicillin-Resistant Staphylococci by Multiplex Polymerase Chain Reaction Assay

Abstract: A multiplex polymerase chain reaction (PCR) assay using oligonucleotide primers to detect mecA and 16S ribosomal RNA gene was developed to aid in identification of methicillin-resistant staphylococci. Validation included 99 isolates of staphylococcus grouped into one of five categories: methicillin-susceptible coagulase-negative staphylococcus (MSCNS), methicillin-resistant coagulase-negative staphylococcus (MRCNS), methicillin-susceptible Staphylococcus aureus (MSSA), high beta-lactamase producing S aureus (HiBSA), and methicillin-resistant S aureus (MRSA). mecA was detected in MRSA (21/21), and in MRCNS (20/20), but not in MSSA (0/20). mecA was occasionally detected in HiBSA (1/19) and MSCNS (3/19). This multiplex PCR assay was also used to test 30 clinical isolates of coagulase-negative staphylococci with discrepancies between results of in vitro tests for susceptibility to oxacillin and was found to be valuable when a more definitive determination of intrinsic methicillin-resistance was desired.

Typing of porcine reproductive and respiratory syndrome viruses by a multiplex PCR assay.

Abstract: A rapid multiplex PCR assay was developed to distinguish between North American and European genotypes of porcine reproductive and respiratory syndrome (PRRS) virus after a portion of the polymerase gene (open reading frame 1b) was sequenced for two North American PRRS virus strains. DNA products with unique sizes characteristic of each genotype were obtained.

PCR assay based on a microsatellite-containing locus for detection and quantification of Epichloë endophytes in grass tissue.

Abstract: A PCR assay which allows detection and quantification of Epichloe endophytes in tissues of the grass Bromus erectus is described. PCR with specific primers flanking a microsatellite-containing locus (MS primers) amplified fragments 300 to 400 bp in length from as little as 1.0 pg of fungal genomic DNA in 100 ng of DNA from infected plant material. When annealing temperatures were optimized, all Epichloe and Acremonium strains tested, representing many of the known taxonomic groups, yielded an amplification product, indicating that the MS primers may be useful for in planta detection of a variety of related species, including agronomically important Acremonium coenophialum and Acremonium lolii. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagnostic applications, a B. erectus-specific primer pair was designed for use in multiplex PCR to allow simultaneous amplification of plant and fungal DNA sequences, providing an internal control for PCR failure caused by inhibitory plant compounds present in DNA extracts. For quantitative applications, a heterologous control template in primer binding sites complementary to the MS primers was constructed for use in competitive PCR, allowing direct quantification of Epichloe in plant DNA extracts. The fungal DNA present in infected leaves of B. erectus between 1 and 20 pg per 100 ng of leaf DNA, but the amounts of fungal DNA present in the sheath and blade of a given leaf were correlated, indicating that the degree of infection varied between plant individuals but that leaves were colonized in a uniform way.

Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes

Abstract: We describe a polymerase chain reaction (PCR) that allowed detection of rRNA consensus sequences from the DNA extracted from a wide range of bacterial species in amounts as low as 10 fg. To avoid false positive results with universal primers for 16S rRNA PCR, contaminating DNA had to be eliminated from the polymerase preparations. Decontamination was undertaken before PCR to optimize treatment with DNaseI, and was followed by DNase inactivation at 94 degrees C for 50 min, which eliminated contaminating DNA at concentrations of up to 100 pg. After optimization of PCR conditions for each polymerase. Deep Vent Exo-polymerase (New England Biolabs, Beverly, MA), and super-Taq polymerase (HT Biotechnology, Cambridge, UK) were more effective than Ampli-Taq polymerase (Perkin-Elmer Cetus, Norwalk CT), Ampli-Taq LD polymerase (Perkin-Elmer Cetus) or Deep-vent polymerase (New England Biolabs). The technique described in this article might prove to be a universal method for PCR detection of small numbers of unidentified bacteria in usually sterile clinical sites, such as blood and cerebrospinal fluids, in which a broad spectrum of pathogens can be expected.

Amplification of anonymous DNA fragments using pairs of long primers generates reproducible DNA fingerprints that are sensitive to genetic variation

Abstract: The reproducibility and potential applications of anonymous amplification protocols can be improved by using pairs of primers, each of 18 to 24 bases, to replace the single 8 to 10 base primers normally used in randomly amplified polymorphic DNA (RAPD) or DNA amplification fingerprinting (DAF) methods. Amplification using large primer pairs (LP-RAPD) generates 5 to 30 bands that can be resolved on standard agarose gels. Complex fingerprints can be readily generated from viruses, bacteria, fungi, plants, invertebrates and vertebrates. We also present evidence that a number of polymerase chain reaction (PCR) methods, including those based on the use of enterobacterial repetitive intergenic consensus (ERIC-PCR) or microsatellite primed (MP-PCR) sequence, may in essence operate by the same mechanism as LP-RAPD. Using standard LP-RAPD protocols, reproducible fingerprints can be generated from a single specimen using different thermocyclers, regardless of the mechanism used for thermocycling (air-cooled, Peltier effect, or robotic arm). LP-RAPD is sensitive to intraspecific and interspecific genetic variation, demonstrated here by analysis of mites and apple cultivars. Approximately 50% of LP-RAPD products are expected to have different primers at either end. Polymorphic bands with this arrangement can be recovered from the gel and directly sequenced using the LP-RAPD primers themselves. The efficiency of sequencing is improved by the length of the LP-RAPD primers. This method has the potential to allow the production of allele-specific species markers in less than two days.

Multiplex PCR assay for immediate identification of the infecting species in patients with mycobacterial disease.

Abstract: Rapid identification of infecting mycobacterial species enables appropriate medical care decisions to be made. Our aim was to demonstrate the clinical usefulness of the multiplex PCR assay, a test based on PCR, which permits direct identification of 12 mycobacterial species in clinical specimens. A total of 259 specimens from 177 patients who had clinical symptoms of mycobacterial disease but for whom there were difficulties in diagnosis were tested. Specimens were analyzed within 48 h of receipt of the sample. Mycobacteria were identified in 102 specimens; 66 specimens contained nontuberculous mycobacteria, and 36 specimens contained Mycobacterium tuberculosis complex mycobacteria. The PCR assay identified the mycobacterial species in 43 (97.7%) of 44 microscopy- and culture-positive specimens and in 15 (93.8%) of 16 culture-positive, microscopy-negative specimens. It also permitted species identification in infections caused by more than one mycobacterial species. For 56 (96.5%) of the 58 specimens from patients with infections caused by opportunistic mycobacteria, the organisms were identified with the PCR assay. The test was useful also for the identification of fastidious mycobacteria, e.g., M. genavense, and those that cannot be cultured, e.g., M. leprae. After resolution of discrepant results, the sensitivity of the PCR assay was 97.9%, the specificity was 96.9%, the positive predictive value was 95.0%, and the negative predictive value was 98.7%. For culture these values were 60.8, 100, 100, and 81.0%, respectively. Thus, the multiplex PCR assay enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.