Analytic performance and contamination control methods of a ligase chain reaction DNA amplification assay for detection of Chlamydia trachomatis in urogenital specimens

Hu, Hsiang-Yun; Burczak, John D.; Leckie, Gregor; Ray, Keith A.; Muldoon, Sharon; Lee, Helen H.

doi:10.1016/0732-8893(95)00272-3

Cited by 10 publications

(5 citation statements)

References 19 publications

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“…The system described in this report includes several measures that control for contamination, such as the use of a single reaction vessel, which does not require opening after amplification, and the automatic chemical destruction of the amplified product after detection. These measures were previously shown to be extremely effective (1,21). Of the 751 HIV-negative samples tested by the HIV-1/2 qualitative RNA assay, three samples tested positive initially, resulting in an initial frequency of false positivity of 0.4%.…”

Section: Discussionmentioning

confidence: 99%

Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2

et al. 2000

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Early detection of human immunodeficiency virus (HIV) in blood and blood products can be achieved by a sensitive nucleic acid amplification-based assay. We report on the performance of a PCR-based qualitative assay that detects both HIV type 1 (HIV-1) and HIV-2 with a sensitivity of 20 to 50 copies/ml. The assay has a specificity of 99.6% and an inhibition rate of 1.7%. One milliliter of sample is processed with a manifold system and Qiagen columns, and one-third of the extracted sample is used for PCR amplification. An internal control sequence, which is processed and amplified with each sample, monitors for amplification inhibition. Samples are reverse transcribed and are then amplified by reverse transcription-coupled PCR, after which HIV-1- and HIV-2-specific probes are hybridized to the amplified products. Following hybridization, samples are detected in the LCx instrument by microparticle enzyme immunoassay techniques. The detection system has an automated inactivation step that controls for PCR contamination. The HIV-1/2 qualitative RNA assay detects HIV-1 group M subtypes A, B, C, D, E, F, and G and group O. Testing of several HIV-1 seroconversion panels has demonstrated that the HIV-1/2 qualitative RNA assay detects HIV infection on the average of 6 days before p24 antigen can be detected and 11 days before antibodies can be detected.

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Section: Discussionmentioning

confidence: 99%

Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2

et al. 2000

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“…Once the gap is filled, ligase can covalently join the pair of probes to form an amplification product that is complementary to the original target sequence and can itself serve as a target in subsequent rounds of amplification. Detection of Chlamydia trachomatis DNA in clinical samples, including male and female urine, by LCR has been favorably utilized (14,23), and recently, in parallel to our study, the LCR approach has been used to detect M. tuberculosis in respiratory specimens (3). The aim of our study was to establish whether this test is useful for direct detection of the M. tuberculosis complex in clinical samples in patients in Norway.…”

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confidence: 80%

Direct detection of Mycobacterium tuberculosis complex in clinical samples from patients in Norway by ligase chain reaction

et al. 1997

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Our aim was to investigate the use of DNA amplification with the ligase chain reaction (LCR) for detection of the Mycobacterium tuberculosis complex directly in human clinical specimens. The LCR assay employed was the Abbott LCx MTB Assay, which uses the gene encoding protein antigen b as the target template. Four hundred eighty-two samples from 457 patients in one clinical microbiology laboratory in Norway were processed by routine culture analysis (BACTEC culture), direct microscopy (Ziehl-Neelsen staining) and LCR. Of the 118 specimens containing cultivable M. tuberculosis, 106 (90.6%) were detected by LCR. Among the 364 culture-negative specimens, 356 samples were negative also by LCR and 8 (1.6%) were positive by LCR. In five of the eight LCR-positive and culture-negative samples, another sample from the same patient was M. tuberculosis culture positive and/or the patient had symptoms of tuberculosis. In comparison with culture, the sensitivity of LCR was 96.7% for smear-positive samples and 72.0% for smear-negative samples, respectively. For all samples combined, the sensitivity, specificity, and positive and negative predictive values were 90.2, 99.2, 97.4, and 96.7%, respectively. Challenging the M. tuberculosis LCR test with DNAs and cultures from strains of Mycobacterium ulcerans and Mycobacterium marinum, which are the mycobacterial species most closely related to the M. tuberculosis complex, resulted in all-negative test results. The sensitivity, specificity, and positive and negative predictive values of BACTEC culture in comparison with the LCR test and clinical criteria were 95.9, 100, 100, and 98.6%, respectively. A certain prioritization of samples subjected to the LCR assay should be based on clinical indications and risks with regard to infection transmission and patient isolation policy. More automation and lower expenses are generally desired for nucleic acid amplification kits. However, this M. tuberculosis LCR assay represents a valuable tool in routine mycobacterial diagnostics.

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“…Specimens were considered gap LCR positive if their sample-to-cutoff (S/CO) ratios were Ն1.0. After detection, the LCx analyzer chemically inactivated the amplification product (17).…”

Section: Methodsmentioning

confidence: 99%

“…A more sensitive version of LCR, called gap LCR, requires thermostable DNA polymerase in addition to thermostable DNA ligase (4,8). Gap LCR assays have been developed on the automated LCx system for the specific detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycobacterium tuberculosis (7,10,11,13,17).…”

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Method for Reduction of Inhibition in a Mycobacterium tuberculosis -Specific Ligase Chain Reaction DNA Amplification Assay

Leckie

Erickson

et al. 1998

J Clin Microbiol

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The present study describes the identification of inhibitors of aMycobacterium tuberculosis-specific gap ligase chain reaction (LCR) DNA amplification assay as well as a method for their removal. A major contributor to inhibition was deduced to be a calcium phosphate precipitate, CaHPO4. The precipitate forms duringN-acetyl-l-cysteine–sodium hydroxide (NALC-NaOH) decontamination, digestion, and concentration of respiratory specimens. The solubility product of CaHPO4 precipitate at pH 7.8, the pH at which gap LCR is optimized, indicates that the precipitate releases an amount of phosphate ions sufficient to inhibit amplification. A method for removal of the precipitate was identified. The precipitate is dissociated by exposing it to a mildly acidic (pH 4.1) buffer during the first of two centrifugation steps; the inhibitory phosphate ions are removed by the centrifugation steps. When 100 NALC-NaOH respiratory sediments were tested by gap LCR, none of the sediments were inhibitory when the acidic buffer was used while 24 samples were inhibitory when TE buffer, pH 7.8, was used. In another study, when the acidic buffer wash was applied to 1,440 NALC-NaOH respiratory sediments, only 10 sediments were found to be inhibitory. None of the inhibited sediments were culture positive for M. tuberculosis. This work demonstrates that when inhibition mechanisms are identified, relatively simple protocols can be used to obtain low inhibition rates and to allow the use of larger volume equivalents in amplification reactions.

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Analytic performance and contamination control methods of a ligase chain reaction DNA amplification assay for detection of Chlamydia trachomatis in urogenital specimens

Cited by 10 publications

References 19 publications

Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2

Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2

Direct detection of Mycobacterium tuberculosis complex in clinical samples from patients in Norway by ligase chain reaction

Method for Reduction of Inhibition in a Mycobacterium tuberculosis -Specific Ligase Chain Reaction DNA Amplification Assay

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