scholarly journals Evaluation of AMPLICOR Neisseria gonorrhoeae PCR Using cppB Nested PCR and 16S rRNA PCR

1999 ◽  
Vol 37 (2) ◽  
pp. 386-390 ◽  
Author(s):  
David J. Farrell
Keyword(s):  
16S Rrna ◽  
Neisseria Gonorrhoeae ◽  
False Positive ◽  
Vaginal Swab ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Diagnostic Systems ◽  
Pcr Method ◽  
Neisseria Subflava ◽  
Positive Results

Certain strains of Neisseria subflava andNeisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence (∼9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICORN. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

scholarly journals Utility of a PCR-based method for rapid and specific detection of toxigenic Microcystis spp. in farm ponds

2020 ◽  
Vol 32 (3) ◽  
pp. 369-381 ◽  
Author(s):  
Jian Yuan ◽  
Hyun-Joong Kim ◽  
Christopher T. Filstrup ◽  
Baoqing Guo ◽  
Paula Imerman ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Low Cost ◽  
Farm Animals ◽  
Rrna Gene ◽  
Analytical Sensitivity ◽  
Water Safety ◽  
Farm Ponds ◽  
Pcr Method

Microcystis is a widespread freshwater cyanobacterium that can produce microcystin, a potent hepatotoxin harmful to animals and humans. Therefore, it is crucial to monitor for the presence of toxigenic Microcystis spp. to provide early warning of potential microcystin contamination. Microscopy, which has been used traditionally to identify Microcystis spp., cannot differentiate toxigenic from non-toxigenic Microcystis. We developed a PCR-based method to detect toxigenic Microcystis spp. based on detection of the microcystin synthetase C ( mcyC) gene and 16S rRNA gene. Specificity was validated against toxic and nontoxic M. aeruginosa strains, as well as 4 intergeneric freshwater cyanobacterial strains. Analytical sensitivity was as low as 747 fg/µL genomic DNA (or 3 cells/µL) for toxic M. aeruginosa. Furthermore, we tested 60 water samples from 4 farm ponds providing drinking water to swine facilities in the midwestern United States using this method. Although all water samples were positive for Microcystis spp. (i.e., 16S rRNA gene), toxigenic Microcystis spp. were detected in only 34 samples (57%). Seventeen water samples contained microcystin (0.1–9.1 μg/L) determined with liquid chromatography–mass spectrometry, of which 14 samples (82%) were positive for mcyC. A significant correlation was found between the presence of toxigenic Microcystis spp. and microcystin in water samples ( p = 0.0004). Our PCR method can be a low-cost molecular tool for rapid and specific identification of toxigenic Microcystis spp. in farm ponds, improving detection of microcystin contamination, and ensuring water safety for farm animals.

scholarly journals Design and Validation of Transcription-Mediated-Amplification Nucleic Acid Amplification Tests forMycoplasma genitalium

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Brett Kirkconnell ◽  
Barbara Weinbaum ◽  
Katherine Santos ◽  
Trisha Le Nguyen ◽  
Bryan Vinluan ◽  
...  
Keyword(s):  
Clinical Performance ◽  
Mycoplasma Genitalium ◽  
Vaginal Swab ◽  
Nucleic Acid Amplification ◽  
Clinical Validation ◽  
Analytical Sensitivity ◽  
Reference Standard ◽  
Content Type ◽  
Sensitivity Specificity

ABSTRACTMycoplasma genitaliumis a sexually transmitted bacterium linked to adverse sexual and reproductive health outcomes in women and men.M. genitaliumis difficult to culture, and in the absence of validated amplified molecular methods for diagnosis of infection, there is no reference standard available for use as a comparator for the validation of newM. genitaliumdiagnostic tests. We evaluated the analytical and clinical performance of three transcription-mediated amplification (TMA) tests forM. genitalium, each targeting unique rRNA sequences, for use as a composite comparator for clinical validation of the AptimaMycoplasma genitalium(AMG) assay, anin vitrodiagnostic (IVD) TMA test that targets 16 s rRNA ofM. genitalium. Analytical sensitivity, specificity, and strain inclusivity of all four TMA tests were determined using nine laboratory strains ofM. genitaliumand 56 nontarget bacteria, protozoa, and viruses. Analytical sensitivity of the tests forM. genitaliumranged from 0.017 to 0.040 genome equivalents/ml. None of the nontarget organisms evaluated cross-reacted with any test. A composite comparator reference standard consisting of the 3 alternate (Alt) TMA tests was used to evaluate the clinical performance of the AMG assay by testing residual vaginal swab, female urine, and male urine specimens obtained from 1,400 adult subjects from three U.S. clinical sites. Using this reference standard to establish infected specimen status, the sensitivity, specificity, and overall agreement of the AMG IVD assay were 100%, 99.9%, and 99.9%, respectively. These results demonstrate the utility of molecular composite reference standard methodology for the clinical validation of future IVD tests for this organism.

Comparison of the Nucleic Acid Amplification System NASBA® and Agar Isolation for Detection of Pathogenic Campylobacters in Naturally Contaminated Poultry

1996 ◽  
Vol 59 (7) ◽  
pp. 683-687 ◽  
Author(s):  
M. UYTTENDAELE ◽  
R. SCHUKKINK ◽  
B. VAN GEMEN ◽  
J. DEBEVERE
Keyword(s):  
Nucleic Acid ◽  
False Positive ◽  
Detection System ◽  
False Negative ◽  
Isolation Procedure ◽  
Nucleic Acid Amplification ◽  
True Nature ◽  
Selective Enrichment ◽  
Amplification System ◽  
Positive Results

A total of 160 poultry products were examined for the presence of pathogenic campylobacters using the traditional agar isolation method and the nucleic acid amplification system NASBA®, both after a 24-h selective enrichment. Pathogenic campylobacters could be isolated from 92 of 160 (57.5%) samples using agar isolation, among which 79 (49.37%) were identified as Campylobacter jejuni, six (3.75%) as C. coli, five (3.12%) as C. lari, and two (1.25%) as unclassified. The NASBA® assay provides a specific and sensitive method for detection of these campylobacters. A total of 149 samples (93.12%) gave similar results for both the traditional isolation procedure on modified Campylobacter charcoal desoxycholate agar and the NASBA® enzyme-linked gel assay detection system. Two false-negative results were obtained with the agar isolation procedure. Nine false-positive results were reported when the NASBA® system was used. However, the high sensitivity of the NASBA® method and indications that in some cases the traditional isolation procedure failed (abundance of a contaminating noncampylobacter bacteria which grew on the Campylobacter selective media) raises doubt about the true nature of these false-positive results. The NASBA® detection assay offers a rapid and useful analytical method when screening for the presence of pathogenic campylobacters. The complete procedure, including 24 h of selective enrichment, required 32 h.

The16S rRNAgene-based PCR method used for the detection of segmented filamentous bacteria in the intestinal microbiota generates false-positive results

Apmis ◽  
2017 ◽  
Vol 125 (10) ◽  
pp. 940-942 ◽  
Author(s):  
Forough L. Nowrouzian ◽  
Liselott Svensson Stadler ◽  
Ingegerd Adlerberth ◽  
Agnes E. Wold
Keyword(s):  
Intestinal Microbiota ◽  
False Positive ◽  
Filamentous Bacteria ◽  
Segmented Filamentous Bacteria ◽  
Pcr Method ◽  
Positive Results

Highly sensitive qualitative methods for serum choriogonadotropin (hCG): clinical specificity studies.

1987 ◽  
Vol 33 (5) ◽  
pp. 677-681 ◽  
Author(s):  
Z L Bandi ◽  
I Schoen ◽  
M DeLara
Keyword(s):  
False Positive ◽  
Quantitative Methods ◽  
Analytical Sensitivity ◽  
Negative Results ◽  
Beta Hcg ◽  
Highly Sensitive ◽  
Menopausal Women ◽  
Pregnancy Testing ◽  
Post Menopausal ◽  
Positive Results

Abstract We screened six highly sensitive kits, designed for serum pregnancy tests, for false-positive results. The two best were then evaluated more extensively. The "BETA-hCG MAIA-clone" (I) and the "TANDEM ICON" (II) kits gave only negative results for 100 sera from men at 5, 10, and 25 int. units/L (1st IRP). Of 100 serum specimens from post-menopausal women three and 10 were hCG positive by the II and the I reagents, respectively, but only at the 5 int. units (1st IRP) of hCG per liter level of sensitivity. At 10 and 25 int. units/L, all specimens were negative by both tests. The manufacturers of these kits recommend pregnancy testing only at the 25 int. units/L level of analytical sensitivity. By quantitative methods, hCG concentrations in the II positive samples ranged from 6 to 20 int. units/L (1st IRP) and lutropin concentrations were between 130 to greater than 150 int. units/L. The medical records of the corresponding patients did not support the presence of trophoblast or any other hCG-secreting tissues. During 15 months of routine use of the II reagents at an analytical sensitivity of 25 int. units/L (1st IRP) hCG for pregnancy testing (greater than 4000 serum specimens from pre-menopausal women), the staff has not reported to us any suspected false-positive findings.

The contribution of APTIMA Combo 2 assay to the diagnosis of gonorrhoea in genitourinary medicine setting

2007 ◽  
Vol 18 (8) ◽  
pp. 551-554 ◽  
Author(s):  
S Moss ◽  
H Mallinson
Keyword(s):  
Nucleic Acid ◽  
Neisseria Gonorrhoeae ◽  
False Positive ◽  
Nucleic Acid Amplification ◽  
Multiple Site ◽  
Site Testing ◽  
Number Of Patients ◽  
Male Patients ◽  
Genitourinary Medicine ◽  
Conventional Microscopy

For 929 female and 821 male patients attending a genitourinary clinic, samples intended for chlamydia diagnosis were dual tested by nucleic acid amplification for both chlamydia and Neisseria gonorrhoeae (NG). The assay used, Gen-probe APTIMA Combo 2 (AC2) detected all cases of NG found by conventional microscopy and culture. AC2 identified additional patients who had partners with NG, but were themselves negative by microscopy and culture. Few, if any, false-positive AC2 results were found. Use of AC2 increased the number of patients treated for NG. It can reduce the number of specimens required and may limit the need for multiple site testing.

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