scholarly journals Method for Reduction of Inhibition in aMycobacterium tuberculosis-Specific Ligase Chain Reaction DNA Amplification Assay

1998 ◽  
Vol 36 (3) ◽  
pp. 764-767 ◽  
Author(s):  
Gregor W. Leckie ◽  
Dwight D. Erickson ◽  
Qizhi He ◽  
Ingrid E. Facey ◽  
Bor-Chian Lin ◽  
...  
Keyword(s):  
Solubility Product ◽  
Dna Amplification ◽  
Acidic Ph ◽  
Major Contributor ◽  
Chain Reaction ◽  
Ligase Chain Reaction ◽  
Phosphate Ions ◽  
Amplification Assay ◽  
Culture Positive ◽  
Respiratory Specimens

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.

scholarly journals Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens

2004 ◽  
Vol 37 (6) ◽  
pp. 431-435 ◽  
Author(s):  
Fabíola Karla Corrêa Ribeiro ◽  
Valdério do Valle Dettoni ◽  
Renata Lyrio Peres ◽  
Solange Alves Vinhas ◽  
Tatiana Resende Có ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Predictive Value ◽  
Direct Detection ◽  
Dna Amplification ◽  
Final Diagnosis ◽  
Chain Reaction ◽  
Ligase Chain Reaction ◽  
Amplification Method ◽  
Sensitivity Specificity ◽  
Respiratory Specimens

A ligase chain reaction DNA amplification method for direct detection of Mycobacterium tuberculosis (Abbott LCx MTB) in respiratory specimens was evaluated. Results from LCx MTB Assay were compared with those from acid fast bacilli smear, culture, and final clinical diagnosis for each patient. A total of 297 respiratory specimens (sputum and bronchial lavage) from 193 patients were tested. The sensitivity, specificity, positive predictive value and negative predictive value of LCx vs culture were 92.7%, 93%, 67.8% and 98.7%, respectively. When compared to the clinical final diagnosis, the sensitivity, specificity, PPV and NPV for LCx were 88.9%, 96.8%, 86.5% and 97.4%, respectively. The sensitivity of LCx MTB assay was 75% for smear-negative, culture positive samples. The results indicate that LCx MTB assay is a rapid, simple and valuable technique as a complementary tool for the diagnosis of tuberculosis.

scholarly journals Rapid Diagnosis of Pulmonary Tuberculosis with the LCx Mycobacterium tuberculosis Assay and Comparison with Conventional Diagnostic Techniques

1998 ◽  
Vol 36 (10) ◽  
pp. 3046-3047 ◽  
Author(s):  
Peter Rohner ◽  
Esther I. M. Jahn ◽  
Beatrice Ninet ◽  
Concetta Ionati ◽  
Rainer Weber ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Direct Detection ◽  
Diagnostic Techniques ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Predictive Values ◽  
Amplification Assay ◽  
Sensitivity Specificity ◽  
Culture Positive ◽  
Respiratory Specimens

The LCx MTB amplification assay is a nucleic acid amplification test intended for the direct detection ofMycobacterium tuberculosis complex in respiratory specimens. We evaluated its performance on 2,001 consecutive respiratory specimens; 78 were culture positive for M. tuberculosis. Sensitivity, specificity, and positive and negative predictive values of this assay for all specimens compared to culture results were 88.5, 97.7, 60.5, and 99.5%, respectively. When referred to resolved clinical diagnosis of active tuberculosis, these values improved to 90.2, 98.4, 72.8, and 99.5%, respectively.

Evaluation of Ligase Chain Reaction for Direct Detection of Mycobacterium tuberculosis in Respiratory Specimens

1998 ◽  
Vol 288 (1) ◽  
pp. 59-65 ◽  
Author(s):  
O. Denis ◽  
J.M. Devaster ◽  
O. Vandenberg ◽  
H. Vanachter ◽  
T. Lafontaine ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Direct Detection ◽  
Chain Reaction ◽  
Ligase Chain Reaction ◽  
Respiratory Specimens

scholarly journals Use of Ligase Chain Reaction with Urine versus Cervical Culture for Detection of Chlamydia trachomatis in an Asymptomatic Military Population of Pregnant and Nonpregnant Females Attending Papanicolaou Smear Clinics

1998 ◽  
Vol 36 (5) ◽  
pp. 1300-1304 ◽  
Author(s):  
Charlotte A. Gaydos ◽  
M. Rene Howell ◽  
Thomas C. Quinn ◽  
Joel C. Gaydos ◽  
Kelly T. McKee
Keyword(s):  
Chlamydia Trachomatis ◽  
Pregnant Women ◽  
Fluorescent Antibody ◽  
Chain Reaction ◽  
Military Population ◽  
Predictive Values ◽  
Papanicolaou Smear ◽  
Ligase Chain Reaction ◽  
Culture Positive ◽  
Urine Specimens

Ligase chain reaction (LCR) (Abbott Laboratories, Abbott Park, Ill.) with first-catch urine specimens was used to detectChlamydia trachomatis infections in 465 asymptomatic military women attending clinics for routine Papanicolaou smear tests. Results were compared to results of cervical culture to determine the sensitivity of the urine LCR and the possible presence of inhibitors of amplification in pregnant and nonpregnant women. Discrepant results for LCR and culture were resolved by direct fluorescent antibody staining of culture sediments, two different PCR assays, and LCR for the outer membrane protein 1 gene. The prevalence of Chlamydia in specimens by urine LCR was 7.3% compared to 5% by culture. For 434 women with matching specimens, there were 11 more specimens positive by LCR than were positive by culture, of which all but one were determined to be true positives. There were four culture-positive, LCR-negative specimens, all from nonpregnant women. The sensitivity, specificity, and positive and negative predictive values of urine LCR after discrepant results were resolved were 88.6, 99.7, 96.9, and 99.0%, respectively. The sensitivity of culture was 71.4%. From the 148 pregnant women (prevalence by LCR, 6.8%), there were no patients who were cervical culture positive and urine LCR negative to indicate the presence in pregnant women of inhibitors of LCR. Additionally, a subset of 55 of the LCR-negative frozen urine specimens from pregnant women that had been previously processed in LCR buffer were inoculated with 5 cell culture inclusion forming units of C. trachomatis each and retested by LCR; all tested positive, indicating the absence of inhibitors of LCR in urine from these pregnant women. The use of LCR testing of urine specimens from asymptomatic women, whether pregnant or not, offers a sensitive and easy method to detect C. trachomatis infection in women.

The presence of a 5′-abasic lesion enhances discrimination of single nucleotide polymorphisms while inducing an isothermal ligase chain reaction

The Analyst ◽  
2016 ◽  
Vol 141 (14) ◽  
pp. 4272-4277 ◽  
Author(s):  
Abu Kausar ◽  
Eiman A. Osman ◽  
Tendai Gadzikwa ◽  
Julianne M. Gibbs-Davis
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Dna Amplification ◽  
Nucleotide Polymorphisms ◽  
Chain Reaction ◽  
Single Nucleotide ◽  
Ligase Chain Reaction

Lesion-induced DNA amplification (LIDA) has been employed in the detection of single nucleotide polymorphisms (SNPs).

scholarly journals Detection and Identification of Mycobacterium tuberculosis Directly from Sputum Sediments by Ligase Chain Reaction

1998 ◽  
Vol 36 (4) ◽  
pp. 1028-1031 ◽  
Author(s):  
Douglas F. Moore ◽  
Janis I. Curry
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Predictive Value ◽  
Positive Culture ◽  
Acid Fast Bacillus ◽  
Chain Reaction ◽  
Ligase Chain Reaction ◽  
Culture Techniques ◽  
Detection And Identification ◽  
Sensitivity Specificity ◽  
Culture Positive

Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a ligase chain reaction (LCR)-based assay and acid-fast stain and culture techniques. Results from the LCR assay (Abbott LCx Mycobacterium tuberculosis[MTB] Assay) were compared to results from standard culture techniques held for 6 weeks. Four hundred ninety-three specimens from 205 patients suspected of pulmonary tuberculosis were included in the prospective study. Thirty-four (6.9%) of the specimens were culture positive for M. tuberculosis, and 13 (38%) of these were also fluorochrome stain positive. LCR sensitivities and specificities compared to culture were 74 and 98%, respectively. LCR sensitivity was 100% for fluorochrome stain-positive specimens and 57% for fluorochrome stain-negative specimens. Nine LCR-negative, culture-positive specimens were the result of low concentrations ofM. tuberculosis. No inhibitors were detected in any of these specimens. Of the eight LCR-positive, culture-negative specimens, five were from patients with active tuberculosis. With these considered culture misses, final LCR sensitivity, specificity, positive predictive value, and negative predictive value were 77, 99, 91, and 98%, respectively. The same performance values for the fluorochrome acid-fast bacillus smear were 33, 98, 62, and 94%, respectively. After normal laboratory sputum processing, the Abbott LCx MTB Assay can be completed in 6 h. Thus, it is possible to have results available within 8 h of specimen submission.

scholarly journals Evaluation of the illumigene Mycoplasma Direct DNA Amplification Assay

2018 ◽  
Vol 56 (7) ◽  
Author(s):  
Neena Kanwar ◽  
Morgan A. Pence ◽  
Donna Mayne ◽  
Jeffrey Michael ◽  
Rangaraj Selvarangan
Keyword(s):  
Direct Detection ◽  
False Negative ◽  
Reference Method ◽  
Dna Amplification ◽  
The United States ◽  
Community Acquired Pneumonia ◽  
Content Type ◽  
Amplification Assay ◽  
Respiratory Specimens

ABSTRACT Mycoplasma pneumoniae is a common cause of community-acquired pneumonia. The illumigene Mycoplasma Direct (iMD) DNA amplification assay is a qualitative in vitro test utilizing loop-mediated isothermal amplification (LAMP) technology for the direct detection of M. pneumoniae DNA in respiratory specimens. The iMD assay does not require the preextraction of nucleic acids from specimens, which is a prerequisite step for the previously approved illumigene Mycoplasma (iM) assay. The aim of this prospective multicenter study was to evaluate the performance characteristics of the newly developed iMD assay, compared with the iM assay. Subjects with symptoms of upper respiratory illnesses suggesting M. pneumoniae infection were enrolled at three sites in the United States. Respiratory specimens were obtained using dual throat swabs. One swab was tested with the iMD assay at each enrollment site. Reference testing with the iM assay was performed by the manufacturer. Among 456 specimens tested, the iM reference method detected M. pneumoniae in 25 specimens (5.5%), while the iMD assay identified 34 specimens (7.5%) as M. pneumoniae positive. There were 10 false-positive results and 1 false-negative result with the iMD assay. The overall positive and negative agreement rates were 96.0% (95% confidence interval [CI], 80.5 to 99.3%) and 97.7% (95% CI, 95.8 to 98.7%), respectively. The overall agreement rate was determined to be 97.6% (95% CI, 95.7 to 98.6%). We conclude that the iMD test results were comparable to the iM assay results. The removal of the DNA extraction step for the iMD assay simplifies testing, saves time, and reduces the costs of detecting M. pneumoniae from throat swabs, compared to the iM assay.

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