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At least 145 records · Page 8Linked to original sources

PubMed1999

Absolute quantitation of MDR1 transcripts using heterologous DNA standards--validation of the competitive RT-PCR (CRT-PCR) approach.

The multidrug resistance (MDR1) gene product, P-glycoprotein (Pgp), is a 170-kDa ATP-dependent pump that expels a variety of anticancer drugs out of malignant cells, reducing drug accumulation and thus antitumor activity. In recent years, considerable data has been presented that indicates the need to standardize detection methods for Pgp and MDR1. Reverse transcription (RT)-PCR is one of the most sensitive and specific techniques used to detect MDR1. Nevertheless, there is the need to address working criteria for quantitation by RT-PCR. In this study, we describe a flexible assay used to quantify MDR1 gene expression using heterologous (nonhomologous) standards for use in competitive RT-PCR (CRT-PCR). Our guidelines were to use a RT-PCR quantitation method that was independent of exponential phase kinetics, sensitive (detect low levels of gene measurement in clinical samples) and did not require radiolabel. Furthermore, the method would need to be flexible enough for the user to express quantitation as either the number of cells or amount of cDNA used in CRT-PCR. Using low-stringency amplification, heterologous DNA competitors were constructed for MDR1 and as an internal reference, the ubiquitously expressed human histone variant 3.3 (H3.3). The benefits of this approach are threefold: (i) amplification kinetics of target and competitor molecules are identical, (ii) low-stringency PCR is a simple way of constructing heterologous DNA competitors that do not require special storage conditions and (iii) heterologous competitors avoid the formation of heteroduplex molecules. We conclude that CRT-PCR is an extremely flexible and sensitive assay that can quantify MDR1 based on competitive amplification of a heterologous competitor. This might complement future efforts to standardize MDR1 detection methods using RT-PCR.

Antisense Elements (Genetics)↗

PubMed2006

Detection sensitivity and quantitation of Plasmodium falciparum var gene transcripts by real-time RT-PCR in comparison with conventional RT-PCR.

Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples.

Animals↗

PubMed1997

Validation of HLA-DR locus typing in forensic specimens by combining PCR-SSP with PCR-RFLP.

The applicability of HLA-DR DNA typing combined with PCR-SSP (sequence specific primers) and PCR-RFLP (restriction fragment length polymorphism) to forensic practice was investigated. PCR-SSP was as effective as serological HLA-DR typing in determining DR types. For more precise definition of DRB1 alleles encoding DR2, DR4, and DR8 antigens, which are fairly common in Japan, we used the PCR-RFLP method. For increasing the sensitivity of this system, we used the nested or semi-nested PCR technique. The minimum amount of template DNA needed for typing was 10 ng of genomic DNA in the case of ordinary PCR, whereas 10 pg of DNA was enough in nested and semi-nested PCR. HLA-DR and-DRB1 alleles were able to be determined from the small amounts of DNA available in forensic materials using the PCR-SSP and subsequent PCR-RFLP methods.

Base Sequence↗

PubMed2004

Clinical identification of common species of dermatophytes by PCR and PCR-RFLP.

To find a fast and efficient way of identifying seven common dermatophytes in clinical practice, we used the techniques of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) targeting Topoisomerase II gene. The DNA of 7 dermatophytes, along with Candida albicans, Aspergillus terreus and Aspergillus flavus were amplified by consensus primer dPsD1. They were then subjected to a second PCR with primers dPsD2 and species-specific primers PsT and PsME separately. 6 of the products generated by dPsD2 were digested with restriction enzyme Hinc II. DNA fragments of 3390 bp and 2380 bp was amplified by using consensus primer dPsD1 and dPsD2 from the genomic DNA of each dermatophyte species separately. By combining the results of the two species-specific primer sets (PsT and PsME), all species of dermatophyte yielded unique sizes-set of PCR products expect for T. mentagrophytes and T. tonsurans. From the restriction profiles of Hinc II, 6 of the 7 dermatophytoses were diagnosed to species level including T. mentagrophytes and T. tonsurans. By combining the results of the PCR and PCR-RFLP, the 7 common dermatophytes can be identified to species level. It is conclude that the multiplex PCR and PCR-RFLP identification targeting the DNA topoisomerase II gene is rapid and efficient.

Arthrodermataceae↗

PubMed1999

Fingerprinting of mixed bacterial strains and BIOLOG gram-negative (GN) substrate communities by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR).

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities.

DNA Fingerprinting↗

PubMed2006

Development of allele-specific PCR and RT-PCR assays for clustered resistance genes using a potato late blight resistance transgene as a model.

Members of the NBS-LRR gene family impart resistance to a wide variety of pathogens and are often found clustered within a plant genome. This clustering of homologous sequences can complicate PCR-based characterizations, especially the study of transgenes. We have developed allele-specific PCR and RT-PCR assays for the potato late blight resistance gene RB. Our assay utilizes two approaches toward primer design, allowing discrimination between the RB transgene and both the endogenous RB gene and numerous RB homeologs. First, a reverse primer was designed to take advantage of an indel present in the RB transgene but absent in rb susceptibility alleles, enhancing specificity for the transgene, though not fully discriminating against RB homeologs. Second, a forward primer was designed according to the principles of mismatch amplification mutation assay (MAMA) PCR, targeting SNPs introduced during the cloning of RB. Together, the indel reverse primer and the MAMA forward primer provide an assay that is highly specific for the RB transgene, being capable of distinguishing the transgene from all RB endogenous gene copies and from all RB paralogs in a diverse collection of wild and cultivated potato genotypes. These primers have been successfully multiplexed with primers of an internal control. The multiplexed assay is useful for both PCR and RT-PCR applications. Double MAMA-PCR, in which both PCR primers target separate transgene-specific SNPs, was also tested and shown to be equally specific for the RB transgene. We propose extending the use of MAMA for the characterization of resistance transgenes.

Alleles↗

PubMed1995

Detection of hepatitis C viral sequences in serum by 'nested' polymerase chain reaction (PCR) and a commercial single-round PCR assay.

BACKGROUND: Demonstration of the hepatitis C virus (HCV) genome is usually done with combined reverse transcription and polymerase chain reaction (RT-PCR) employing nested primer sets. Recently, a commercial PCR assay (Amplicor PCR assay), based on a simplified sample preparation procedure, a single, combined reverse transcription and polymerase chain reaction (RT-PCR), and a microwell plate capture and detection, has been developed. OBJECTIVE: The aim of the present study was to compare the new Amplicor assay with an 'in-house' PCR. Additional testing included a third-generation enzyme immunoassay for anti-HCV antibodies, the Wellcozyme HCV Western Blot, which is equivalent to a third-generation recombinant immunoblot assay. Furthermore, HCV genotypes were classified. STUDY DESIGN: Sera from a total of 127 patients were studied. After screening with a third-generation enzyme immunoassay (EIA), the Wellcozyme HCV Western Blot, was performed as well as the conventional RT-PCR and the Amplicor PCR. Specimens, which were found positive by testing with the Amplicor kit, were subjected to storage at room temperature for 96 h. RESULTS: A total of 52 patients were found to be positive for anti-HCV by the third-generation EIA. With the Amplicor assay, the HCV genome was detected in 38 patients. In comparison with the 'in-house' assay, two discrepant results were found. Resolution of discrepant samples increased the total number of true positives to 39. A good correlation was found between a positive anti-HCV test result and the presence of HCV-RNA by RT-PCR. No significant reduction in the amount of amplification product was observed by retesting of suboptimally stored samples with the Amplicor assay. CONCLUSION: Because of the rapidity and the improved ease of handling, the Amplicor assay was found to be a good contribution for detection of HCV in serum.

Journal Article↗

PubMed2004

Routine diagnostics of Lawsonia intracellularis performed by PCR, serological and post mortem examination, with special emphasis on sample preparation methods for PCR.

The aim of this study was to find suitable and reliable tools for demonstrating Lawsonia intracellularis in routine clinical diagnosis. Firstly, a method to prepare tissue samples before a polymerase chain reaction (PCR) was evaluated in pigs submitted for necropsy. Secondly, seven different faecal preparation methods and four different DNA polymerases were tested in single or nested PCR, with co-amplification of a mimic molecule. Thirdly, in selected pigs submitted for necropsy, tissue and faecal samples were examined histopathologically and by PCR, and blood samples were analysed serologically. Detection of L. intracellularis in tissue preparations by PCR showed good specificity and correlated to lesions found at necropsy. The sensitivity in spiked tissue samples was 10(1)-10(2) mimic molecules per tube. In faecal samples, nested PCR on boiled lysate gave the best result with a sensitivity of 10(2)-10(3) mimic molecules per reaction tube. However, because of the time-consuming procedure and the increased risk for contamination, a commercially available kit was preferred for routine diagnoses, despite a somewhat lower detection rate in subclinically infected pigs. In a few cases, the serological results differed from those obtained by PCR and by necropsy but the reason for this is not clear. This study indicates that the best method for diagnosis of acute enteritis in growers is PCR on faecal or tissue samples. To determine the presence of the bacteria in a herd, serology or repeated faecal sampling for PCR from target animals, or both, should be used.

Animals↗

PubMed2001

Detection of growth factor receptor RNA in individual hematopoietic cells by in situ RT-PCR; comparison with RT-PCR.

The ability to detect changes in RNA expression in single cells would greatly enhance understanding of the molecular basis of biological responses to positive and negative growth regulators. To this end, we compared expression of RNA encoding the receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, leukemia inhibitory factor (LIF) and stem cell factor (SCF) in populations of primitive hematopoietic progenitors (lineage marker negative, Lin(-), and Lin(-) c-Kit(+)) by RT-PCR and in situ RT-PCR. Both Lin(-) and Lin(-) c-Kit(+) progenitors expressed all receptors by RT-PCR. However, RT-PCR could not distinguish between the possibility that all cells expressed growth factor receptor RNA, or the possibility that only a proportion of cells expressed RNA. Therefore, we used in situ RT-PCR to examine growth factor receptor mRNA expression in individual cells. In contrast to RT-PCR, we observed that only 40-80% of Lin(-) cells and 75-100% of Lin(-) c-Kit(+) cells were positive for expression of the growth factor receptor subunits, demonstrating that not all cells were receptor positive. We found that in situ RT-PCR could also be used to measure induction or repression of receptor RNA expression in these cell populations. Specifically, the percentage of cells expressing IL-6alpha receptor RNA decreased from 88% positive in freshly harvested cells to 9% in Lin(-) c-Kit(+) cells cultured in IL-3 for 18 h. Thus, in situ RT-PCR can be used to detect and quantify the number of individual cells that express growth factor receptor mRNA, and may also be useful to measure changes in expression of other endogenous genes or genes introduced by transfection and gene therapy vectors.

Actins↗

PubMed2000

Duplex RT-PCR: reagent concentrations at reverse transcription stage affect the PCR performance.

Test conditions for the simultaneous detection of potato leafroll virus (PLRV) and potato virus Y (PVY) in dormant tubers and leaves by reverse transcription-polymerase chain reaction (RT-PCR) were optimized. Various factors optimized at the reverse transcription (RT) stage rather than at the amplification (PCR) stage affected the outcome. In the simplex RT-PCR a onefold dNTPs concentration (0.5 mM) was sufficient in yielding a PLRV or PVY band. In contrast, the duplex RT-PCR required a minimum twofold dNTPs concentration (1.0 mM) during RT to produce distinct bands in PCR. Similarly, various proportions of antisense primers of PLRV and PVY used during RT affected subsequent duplex RT-PCR. Optimal amplification of both viruses were obtained at a ratio of 0.90:0.49 microM of PLRV:PVY antisense primers. An interaction of dNTPs and RNA template concentration was observed. A higher concentration of RNA was required at onefold dNTPs concentration than at twofold dNTPs. Dilutions down to 1:300 of RNA template yielded distinct bands of both viruses at twofold dNTPs concentration. At optimized conditions of the duplex RT-PCR both viruses were reliably detected in composite samples at a ratio of one part infected sap mixed with 399 parts of sap from healthy tubers. Application of optimized conditions to singly- and doubly-infected tubers detected both viruses from naturally infected field-grown tubers. A nearly perfect correlation (r(2)=0.99) was observed between visible plant symptoms and the virus detection from leaves and tubers by the duplex RT-PCR.

DNA Primers↗

PubMed1997

A comparison of RT-PCR, in-situ hybridisation and in-situ RT-PCR for the detection of rhinovirus infection in paraffin sections.

We describe an in-situ RT-PCR method for the amplification of rhinovirus (RV) in fixed, paraffin-embedded HeLa cells employed as a model for human respiratory epithelium. HeLa cells were infected in-vitro with inocula of rhinovirus-16 ranging from 10(2) to 10(6) 50% tissue culture infective doses (TCID50), incubated for 18 h then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to standard RT-PCR, in-situ hybridisation (ISH) or in-situ RT-PCR using specific oligonucleotide primers or probes directed against the 5' non-coding region of RV RNA. RT-PCR was found to be capable of detecting RV16 RNA in one 8 microns-thick section of cells infected with the lowest virus titre. ISH using digoxigenin labelled oligonucleotide probes located RV16 signal in the majority of HeLa cells at the highest virus titre, but in few or no cells with the lowest virus titre. In contrast, in-situ RT-PCR detected RV16 in the majority of cells infected with this amount of RV16. There was a slight loss of morphology and fine localisation associated with the in-situ thermal cycling process. However, the sensitivity of in-situ RT-PCR is comparable to standard RT-PCR and greater than ISH for the detection of RV. In-situ RT-PCR has wide applications for sensitive localization of low copy viral and RNA sequences within cells to investigate the role of viruses in a variety of clinical conditions.

HeLa Cells↗

PubMed1997

Simultaneous detection and typing of plum pox potyvirus (PPV) isolates by heminested-PCR and PCR-ELISA.

Two techniques for simultaneous detection and typing of plum pox potyvirus (PPV) isolates belonging to the D or M serotypes, heminested PCR (H-PCR) and PCR-ELISA, have been developed. Ten PPV isolates typed using PPV-D and PPV-M specific monoclonal antibodies by ELISA-DASI were used to validate these two methods. The results obtained show a complete coincidence of the nucleic acid-based techniques with the serological data. When serial dilutions of infected plant extracts were assayed, H-PCR and PCR-ELISA were found to be 100 times more sensitive than the more conventional immunocapture-PCR (IC-PCR) assay. Testing of 228 PPV-infected fruit tree samples coming from different hosts and locations indicated that so far only PPV type D appears to be present in Spain and in Chile. Coupled with print-capture sample preparation (Olmos et al., Nucl. Acids Res. 24, 2192-2193, 1996) the increased sensitivity provided by heminested-PCR allowed the detection of PPV targets of D and M types, in wingless individuals of the aphid vector Aphis gossypii.

Amino Acid Sequence↗

PubMed1994

Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in-situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in-situ RT-PCR.

To evaluate reliable methods for detection of hepatitis C virus (HCV) infection in routinely processed liver biopsies we analyzed formaldehyde-fixed and paraffin-embedded liver specimens of 10 patients with serological confirmed HCV infection. We compared (1) conventional histology; (2) indirect immunofluorescence using the mAb TORDJI-22 (Clonatec, Paris, France); (3) RT-PCR using total RNA and Southern blotting with chemiluminescent detection; (4) non-radioactive in-situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes; (5) direct in-situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and (6) indirect in-situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. [Gastroenerology 1993;104:595] together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies specimens are adequate; (2) the commercially available mAb TORDJI-22 appears to crossreact with non-HCV epitopes, resulting in false positives; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in-situ RT-PCR or ISH; and (5) direct in-situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. It is concluded, that several molecular methods for HCV detection must await further improvements of protocols to be suitable for routine diagnostics on paraffin-embedded liver biopsies.

Base Sequence↗

PubMed1999

Detection of herpes simplex virus (HSV) genome using polymerase chain reaction (PCR) in clinical samples comparison of PCR with standard laboratory methods for the detection of HSV.

BACKGROUND: Diagnosis of Herpes simplex virus (HSV) infections is achieved by detecting the antigen and isolating the virus from the specimen, which requires 7-28 days. With the recently introduced molecular biological technique of polymerase chain reaction (PCR), the diagnosis of HSV infections has been made more rapid and specific. OBJECTIVE: We evaluated PCR in comparison with the standard laboratory methods on different types of clinical specimens referred to in our laboratory. STUDY DESIGN: A total of 54 specimens, from 54 patients, were investigated. Antigen detection on direct smears was carried out using fluorescent antibody test (FAT) and virus isolation was performed using conventional tube culture method. PCR was carried out with the DNA extracted from various specimens using primers, which coded for the DNA polymerase gene giving a 179 base pair (bp) product. RESULTS: The primers were specific for HSV-1 and HSV-2, and the sensitivity of the primers was found to be 0.5 and 0.2 fg in the detection of HSV-1 and HSV-2 DNA, respectively. Of the 50 specimens (excluding 4 archival formalin fixed tissue specimens, which were not subjected to virological methods of detection) HSV was detected by virological methods and PCR in nine specimens, and by PCR alone in 15 additional specimens, thus increasing the analytical sensitivity significantly by 30% (McNemar test: P = 0.0001). The positivity of PCR in all nine virologically positive specimens and the 4 archival specimens obtained from proven lesions of HSV infections further confirmed the specificity of the PCR. CONCLUSION: PCR, in our study, was found to be a rapid, specific and highly sensitive method for the detection of HSV in clinical specimens.

Animals↗

PubMed2006

Electrochemical genosensor based on peptide nucleic acid-mediated PCR and asymmetric PCR techniques: Electrostatic interactions with a metal cation.

The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 degrees C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The attachment of long single-strands on GCE surface caused the accumulation of [Co(NH3)6]3+ and a high current response. Here, we report a versatile method that would allow for simple and rapid analysis of nucleic acids in combination with PNA-mediated PCR and asymmetric PCR techniques by using an electrochemical genosensor.

Base Sequence↗

PubMed2003

Molecular diagnostics in clinical parasitology and mycology: limits of the current polymerase chain reaction (PCR) assays and interest of the real-time PCR assays.

Polymerase chain reaction (PCR) represents a major breakthrough for the diagnosis of infectious diseases. However, the absence of standardized kits for commercially unattractive targets, such as most of the parasites and the fungi, has led to the development of numerous in-house PCR assays. The performances reported, both for the sensitivity and the specificity of these assays are very divergent. For instance, for the antenatal diagnosis of toxoplasmosis, the sensitivity is either 97.4%, or 64%. For the diagnosis of toxoplasmosis in HIV-positive patients, the PCR on blood is either of limited value with a sensitivity of 13% or of excellent yield with a sensitivity of 87.5%. Similar results are reported for the diagnosis of invasive aspergillosis in bone-marrow-transplant recipients. The patients and the clinical specimens tested are often different. This can explain some of the discrepancies. However, when performed, the quality controls on identical specimens show different results depending on the laboratories. An analysis of the PCR techniques used shows that the control of false positive results as a result of carry-over and false negative results owing to PCR inhibitors is far from being systematic. These shortcomings of 'classical' PCR should be solved when real-time PCR assays are developed, leading to some standardization. Automated DNA extraction should also be useful to achieve this goal. Comparison between laboratories should then be possible and regular quality controls will be necessary to ensure the reliability of real-time PCR assays.

Aspergillosis↗

PubMed2000

TB PCR in the early diagnosis of tuberculous meningitis: evaluation of the Roche semi-automated COBAS Amplicor MTB test with reference to the manual Amplicor MTB PCR test. .

SETTING: Cecilia Makiwane Hospital, Mdantsane, Eastern Cape, Republic of South Africa. OBJECTIVE: To assess the role of the semi-automated Roche COBAS AMPLICOR(TM)Mycobacterium tuberculosis PCR test in the diagnosis of tuberculous meningitis (TBM). DESIGN: Eighty-three specimens of cerebrospinal fluid (CSF) were collected prospectively from 69 patients with suspected TBM. The COBAS AMPLICOR TB PCR test was compared with the manual AMPLICOR(TM)TB PCR test, clinical and cerebrospinal fluid (CSF) findings, direct ZN smear and radiometric TB culture. RESULTS: CSF from 7/40 (17.5%) patients treated for TBM were positive by TB COBAS AMPLICOR(TM). The sensitivity of the test was not significantly different (p=0.375) from the manual TB AMPLICOR(TM)PCR test. The comparative sensitivities of the TB COBAS AMPLICOR(TM)PCR and the manual AMPLICOR PCR for detecting cases of definite and probable TBM from CSF collected within 9 days of commencing antituberculosis treatment were 40% and 60% respectively. All 29 patients not treated for TBM were negative by COBAS AMPLICOR(TM), giving a specificity of 100%. CONCLUSION: The COBAS AMPLICOR(TM)TB PCR test is a rapid and highly specific diagnostic test for TBM. However, there was a non-significant trend favouring slightly greater sensitivity using the manual AMPLICOR(TM)TB PCR test.

Adolescent↗

PubMed1996

Comparison of the amplified Mycobacterium tuberculosis (MTB) direct test, Amplicor MTB PCR, and IS6110-PCR for detection of MTB in respiratory specimens.

Several nucleic acid amplification techniques (NAAT) have been developed for rapid and direct detection of Mycobacterium tuberculosis (MTB) from clinical specimens. This study compared the performances of the Gen-Probe Amplified MTB Direct Test (AMDT), Roche Amplicor MTB PCR test, and an IS6110-PCR assay with acid-fast smear and culture in the detection of MTB from 428 respiratory specimens from 259 patients. Patients' charts were reviewed for clinical correlation. Of 98 specimens that were clinically positive for MTB, acid-fast smear was positive in 50% of cases, culture in 93%, IS6110-PCR in 83%, AMDT in 84%, and Amplicor MTB PCR in 80%. Of 337 specimens that were negative for MTB, 117 (35%) were positive for nontuberculous mycobacteria. Specificities were as follows: smear, 89%; culture, 100%; IS6110-PCR, 99%; AMDT, 98%; and Amplicor MTB PCR, 96%. The accuracies of the tests were 80%, 98%, 96%, and 92%, respectively. MTB culture-positive specimens that were smear-negative were detected by AMDT and IS6110-PCR in 77% of cases and by Amplicor MTB PCR in 70%. NAAT was less sensitive than was culture for detection of MTB, but all these techniques had acceptable accuracy and were completed within hours. NAAT may be useful for rapid screening of respiratory specimens to distinguish MTB from nontuberculous mycobacteria infection in order to isolate patients.

Bronchoalveolar Lavage Fluid↗