scholarly journals Sequence-independent single-primer-amplification (SISPA) as a screening technique for detecting unexpected RNA viral adventitious agents in cell cultures

2021 ◽  
Vol 5 (1) ◽  
pp. 008-012
Author(s):  
Bahador Davood ◽  
Mohammadi Ashraf ◽  
Foroughi Abolhasan ◽  
Alirezaie Behnam
Keyword(s):  
Cell Cultures ◽  
False Positive ◽  
Rna Viruses ◽  
Rna Virus ◽  
Negative Control ◽  
Nucleic Acid Amplification ◽  
Random Amplification ◽  
Single Primer ◽  
Positive Results ◽  
Expression Plasmids

The sequence-independent, single-primer amplification (SISPA) enables the random amplification of nucleic acids, allowing the detection and genome sequencing of different viral agents. This feature of SISPA method provides evidence for application of it in monitoring the presence of adventitious RNA viruses in cell cultures. We evaluated SISPA method for the detection of a challenge RNA virus representing adventitious agent in cell cultures. Besides, by optimizing the SISPA method in our laboratory, we found false-positive results on negative control lanes in electrophoresis gels. To investigate the sources of contamination, false-positive results of SISPA were cloned into Escherichia coli cells, sequenced, and phylogenetically analyzed. This data revealed that the SISPA method can be used as an adjunct method to confirm the absence of unexpected adventitious RNA viruses in cell cultures. The phylogenetic analysis of SISPA contaminant sequences showed that the false-positive results were caused by nucleic acid amplification of commercial cDNA synthesis kit reagents, probably tracing back to expression plasmids and host ribosomal sequences, used for the production of enzymes. Therefore, laboratories using random amplification methods must be constantly aware of the potentials of such contaminations, yielding false-positive results and background noise in the final NGS reads.

scholarly journals False-Positive Results for Mycobacterium celatum with the AccuProbe Mycobacterium tuberculosisComplex Assay

2000 ◽  
Vol 38 (7) ◽  
pp. 2743-2745 ◽  
Author(s):  
Ákos Somoskövi ◽  
Jacqueline E. Hotaling ◽  
Marie Fitzgerald ◽  
Vivian Jonas ◽  
Denise Stasik ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
False Positive ◽  
Negative Control ◽  
Positive Results

Mycobacterium celatum type 1 was found to cross-react in the AccuProbe Mycobacterium tuberculosis complex assay. Subsequently, we found a statistically significant increase in the relative light units with lower temperatures, suggesting that it is necessary to perform this AccuProbe assay at between 60 and 61°C. We also recommend the inclusion of M. celatum type 1 as a negative control.

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.

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 CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification

2020 ◽  
Vol 48 (5) ◽  
pp. e30-e30 ◽  
Author(s):  
Bo Tian ◽  
Gabriel Antonio S Minero ◽  
Jeppe Fock ◽  
Martin Dufva ◽  
Mikkel Fougt Hansen
Keyword(s):  
Nucleic Acid ◽  
False Positive ◽  
Rolling Circle Amplification ◽  
Background Level ◽  
Test Sample ◽  
Negative Control ◽  
Nucleic Acid Amplification ◽  
Rolling Circle ◽  
Nonspecific Amplification ◽  
Negative Controls

Abstract False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.

A Collaborative Study on the Detection of Staphylococcal Enterotoxins in Foods with an Enzyme Immunoassay Kit (TECRA)

1996 ◽  
Vol 59 (4) ◽  
pp. 390-397 ◽  
Author(s):  
C. E. PARK ◽  
D. WARBURTON ◽  
P. J. LAFFEY ◽  
Keyword(s):  
Enzyme Immunoassay ◽  
False Positive ◽  
Collaborative Study ◽  
False Negative ◽  
Negative Control ◽  
Rabbit Serum ◽  
Staphylococcal Enterotoxins ◽  
Repeatability And Reproducibility ◽  
Positive Results ◽  
Control Samples

One of the commercially available enzyme immunoassay kits for the detection of staphylococcal enterotoxins (SEs) in foods, the TECRA screening kit (Bioenterprises Pty. Ltd., Roseville, New South Wales, Australia), has microtiter plates coated with a mixture of antibodies to all of the SEs. A collaborative study was conducted to ascertain whether specificity, sensitivity, repeatability, and reproducibility of the results obtained using this kit would meet food-safety criteria. Thirteen Canadian collaborators participated in this study to analyze both various foods to which 1.0 to 3.0 ng of SE/g of food had been added and negative control samples. In addition, the effect of animal serum in these analyses was examined. The results indicate that all collaborators (100%) were able to detect the minimum toxin levels of 1.0 ng of SEA/g of ham and 1.0 ng of SEB/g of salami and SE or SEs in other samples (chicken, turkey, and cheese) containing 2.0 to 3.0 ng/g, without any false-negative results. With regard to negative control samples, all collaborators obtained correct results except when analyzing two types of food: two collaborators (15%) showed weak false-positive results with salami and all analysts found strong false-positive results with mussels. The problem regarding specificity could be largely corrected by treating the sample with rabbit serum (0.1 volume in 1.0 volume food extract). The repeatability and reproducibility of results from the kit were acceptable.

Critical Evaluation of Impedance Phlebography for the Diagnosis of Deep Vein Thrombosis

1974 ◽  
Vol 31 (02) ◽  
pp. 273-278
Author(s):  
Kenneth K Wu ◽  
John C Hoak ◽  
Robert W Barnes ◽  
Stuart L Frankel
Keyword(s):  
Deep Vein Thrombosis ◽  
False Positive ◽  
False Negative ◽  
Critical Evaluation ◽  
Original Method ◽  
Vein Thrombosis ◽  
Negative Results ◽  
False Negative Results ◽  
Deep Vein ◽  
Positive Results

SummaryIn order to evaluate its daily variability and reliability, impedance phlebography was performed daily or on alternate days on 61 patients with deep vein thrombosis, of whom 47 also had 125I-fibrinogen uptake tests and 22 had radiographic venography. The results showed that impedance phlebography was highly variable and poorly reliable. False positive results were noted in 8 limbs (18%) and false negative results in 3 limbs (7%). Despite its being simple, rapid and noninvasive, its clinical usefulness is doubtful when performed according to the original method.

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