scholarly journals Internal control in PCR for Mycobacterium tuberculosis: usefulness and improvement of the diagnosis

2008 ◽  
Vol 51 (4) ◽  
pp. 485-491 ◽  
Author(s):  
Elizabeth Cortez-Herrera ◽  
Rosa Dea Sperhacke ◽  
Daniela Becker ◽  
Afrânio Kritski ◽  
Arnaldo Zaha ◽  
...  
Keyword(s):  
Public Health ◽  
Mycobacterium Tuberculosis ◽  
Internal Control ◽  
False Negative ◽  
Clinical Results ◽  
Public Health Laboratory ◽  
Negative Results ◽  
False Negative Results ◽  
Diagnostic Fragment ◽  
Pcr Test

The aim of this work was to construct and test a plasmidial Internal Control (IC) to detect the inhibition in the PCR test for M. tuberculosis and also its contribution for a Public Health Laboratory routine. The IC was a 600-bp of DNA linked to a plasmid with the same primer sites, allowing the amplification with the 245-bp diagnostic fragment. The amplification of the positive samples rendered the IC and the diagnostic fragment; instead negative samples only showed the IC. A total of 149 tuberculosis samples were studied and introduced the IC to monitor. Results showed 3.3% of the samples without amplification of the IC, suggesting the inhibition. These samples showed results in accordance with the clinical results. The objective of the IC was to identify the false negative results.

scholarly journals Superiority of MALDI-TOF Mass Spectrometry over Real-Time PCR for SARS-CoV-2 RNA Detection

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 730
Author(s):  
Magda Rybicka ◽  
Ewa Miłosz ◽  
Krzysztof Piotr Bielawski
Keyword(s):  
Mass Spectrometry ◽  
Gold Standard ◽  
Viral Genome ◽  
False Negative ◽  
Rt Pcr ◽  
Rna Detection ◽  
Maldi Tof ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Test

At present, the RT-PCR test remains the gold standard for early diagnosis of SARS-CoV-2. Nevertheless, there is growing evidence demonstrating that this technique may generate false-negative results. Here, we aimed to compare the new mass spectrometry-based assay MassARRAY® SARS-CoV-2 Panel with the RT-PCR diagnostic test approved for clinical use. The study group consisted of 168 suspected patients with symptoms of a respiratory infection. After simultaneous analysis by RT-PCR and mass spectrometry methods, we obtained discordant results for 17 samples (10.12%). Within fifteen samples officially reported as presumptive positive, 13 were positive according to the MS-based assay. Moreover, four samples reported by the officially approved RT-PCR as negative were positive in at least one MS assay. We have successfully demonstrated superior sensitivity of the MS-based assay in SARS-CoV-2 detection, showing that MALDI-TOF MS seems to be ideal for the detection as well as discrimination of mutations within the viral genome.

scholarly journals Fluorometric detection of HIV-1 genome through use of an internal control, inosine-substituted primers, and microtiter plate format

1996 ◽  
Vol 42 (5) ◽  
pp. 696-703 ◽  
Author(s):  
B Gérard ◽  
C Peponnet ◽  
G Brunie ◽  
H Cavé ◽  
E Denamur ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Internal Control ◽  
False Negative ◽  
Enzymatic Reaction ◽  
Pcr Amplification ◽  
Microtiter Plate ◽  
Fluorometric Detection ◽  
Negative Results ◽  
False Negative Results ◽  
Hiv 1

Abstract We describe a PCR-based fluorometric assay for the detection of the HIV-1 genome. This technique consists of a reverse hybridization with oligonucleotide probes covalently coated onto a microtiter plate as a solid support. Several improvements to the PCR amplification and detection steps gave greater sensitivity and specificity for HIV-1 screening and resulted in a convenient and rapid technique. False-positive results were avoided by using uracyl DNA glycosylase. False-negative results from the presence of PCR inhibitors were detected by coamplifying an internal control with the viral sequence. False-negative results from viral genome variability were limited by using two pairs of primers and by incorporating inosine at the primer positions corresponding to viral polymorphic nucleotides. Furthermore, the hybridization buffer and enzymatic reaction were optimized to increase the assay's sensitivity. The sensitivity and specificity of the fluorometric detection were similar to those of radioisotopic oligonucleotide solution hybridization; however, hands-on time was reduced, and the use of radioactivity was eliminated. We have used this technique routinely on 115 samples and obtained 100% specificity and high sensitivity (only one false-negative result) according to viral culture and (or) serological status of the patients.

scholarly journals Reducing False Negative PCR Test for COVID-19

2020 ◽  
Vol 9 (3) ◽  
pp. 408-410
Author(s):  
Fatemeh Bahreini ◽  
Rezvan Najafi ◽  
Razieh Amini ◽  
Salman Khazaei ◽  
Saeid Bashirian
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
False Negative ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Creative Commons ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain ◽  
Pcr Test

As the SARS-CoV-2 (COVID-19) pandemic spreads rapidly, there is need for a diagnostic test with high accuracy to detect infected individuals especially those without symptoms. Real-time polymerase chain reaction (RT-PCR) is a common molecular test for diagnosing SARS-CoV-2. If some factors are not taken into consideration when performing this test, it can have a relatively large number of false negative results. In this article, we discuss important considerations that could lead to false negative test reduction. Key words: • SARS-CoV-2 • COVID-19 • Real time polymerase chain reaction • RT-PCR test • Diagnosis • False negatives • Genetics • Emerging disease   Copyright © 2020 Bahreini et al. Published by Global Health and Education Projects, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0)which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in this journal, is properly cited.

scholarly journals Improving of the Nested PCR for Detection of Bovine Leukemia Virus

2021 ◽  
Vol 83 (3) ◽  
pp. 56-65
Author(s):  
L.M. Ishchenko ◽  
◽  
V.V. Nedosekov ◽  
V.D. Ishchenko ◽  
O.Yu. Kepple ◽  
...  
Keyword(s):  
Internal Control ◽  
Nested Pcr ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
False Negative ◽  
Envelope Gene ◽  
Rt Pcr ◽  
Second Stage ◽  
Negative Results ◽  
False Negative Results

Enzootic bovine leukosis caused by a bovine leukemia virus has a significant economic impact and is reported in World Organization for Animal Health(OIE). Aim. The purpose of our work was to improve the nested polymerase chain reaction (PCR) recommended by the OIE conducting it second-stage in real-time (RT) PCR. Such modification does not require the stage of gel electrophoresis and consequently reduces contamination risks and prevents false positive results. Methods. Primers that are recommended by the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE) were used for the first amplification stage. For the second stage of the proposed modification of nested PCR, the primers and probe were designed based on the alignment of the sequences envelope gene of different isolates of bovine leukemia virus including Ukrainian isolates. Amplification of the internal control was carried out for the second stage to prevent false negative results. Results. Comparative studies of 48 blood samples for bovine leukemia virus identification by a proposed nested RT-PCR, nested PCR recommended by the protocol of the OIE, and RT-PCR were conducted. The sample panel included both positive and negative samples. A 100% match of the results of the bovine leukemia virus presence in nested PCR proposed by the OIE and in our proposed nested RT-PCR was obtained. Comparative analysis of results that were obtained using the RT-PCR and the proposed nested RT-PCR showed that false-negative results in 5 samples and 3 doubtful results that require retesting were obtained by use of RT-PCR. The interpretation of the results using nested RT-PCR is more efficient than RT-PCR since the cycle threshold value of positive samples obtained using RT-PCR was in the range of 24–40 cycles, whereas in the case of nested RT-PCR using, the value of Ct was in the range of 4–20 cycles. Conclusions. Proposed nested PCR modification includes the combination of the OIE recommendation about nested PCR and the reduction of the risk of contamination by conducting the second stage in RT-PCR. Results of approbation of proposed nested RT-PCR give a reason to recommend it for the identification of bovine leukemia virus.

scholarly journals Enhancement of the AMPLICOR Enterovirus PCR Test with a Coprecipitant

1998 ◽  
Vol 36 (11) ◽  
pp. 3408-3409 ◽  
Author(s):  
E. William Taggart ◽  
Carrie L. Byington ◽  
David R. Hillyard ◽  
John E. Robison ◽  
Karen C. Carroll
Keyword(s):  
Test Specimen ◽  
False Negative ◽  
Blind Study ◽  
Specimen Preparation ◽  
Background Signal ◽  
Repeat Testing ◽  
Negative Results ◽  
Data Support ◽  
False Negative Results ◽  
Pcr Test

The incorporation of a commercially available coprecipitant into the AMPLICOR enterovirus PCR test specimen preparation enhanced the sensitivity and reproducibility of this assay. Fifty-five previously tested archived cerebrospinal fluids (CSF) specimens were tested in a blind study in duplicate with and without Pellet Paint coprecipitant (Novagen, Inc., Madison, Wis.). Of these specimens, 26 had previously been determined to be positive and 29 had previously been determined to be negative. All previously positive CSF specimens were positive when Pellet Paint was used and only 18 were positive without Pellet Paint. No previously negative specimens were positive on repeat testing with or without Pellet Paint. The background signal was not affected by the addition of Pellet Paint. These data support the utility of a coprecipitant in minimizing false-negative results.

scholarly journals False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

2020 ◽  
Author(s):  
Marcelo Fruehwirth ◽  
Açucena Veleh Rivas ◽  
Andressa Faria Rahyn Fitz ◽  
Aline Cristiane Cechinel Assing Batista ◽  
Cleypson Vinicius Silveira ◽  
...  
Keyword(s):  
Internal Control ◽  
False Negative ◽  
Rna Extraction ◽  
N Gene ◽  
Gold Standard Method ◽  
Negative Results ◽  
False Negative Results ◽  
Average Variation ◽  
Wrong Diagnosis ◽  
Inhibitor Compounds

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise®). rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80µl of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ < 0.05) delaying Cq between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.80% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

scholarly journals COVID-19 – the challenge to treat a disease and not a positive RT-PCR test

Pharmacia ◽  
2021 ◽  
Vol 68 (1) ◽  
pp. 155-161
Author(s):  
Maria Georgieva Moneva-Sakelarieva ◽  
Yozlem Ali Kobakova ◽  
Petar Yordanov Atanasov ◽  
Danka Petrova Obreshkova ◽  
Stefka Achkova Ivanova ◽  
...  
Keyword(s):  
False Negative ◽  
Rapid Test ◽  
Rt Pcr ◽  
Negative Results ◽  
False Negative Results ◽  
Chest X Ray ◽  
Pulmonary Changes ◽  
Coagulation Status ◽  
Pcr Test

The new pandemic disease COVID is quick spread worldwide.The primary method used for diagnosing of COVID-19 is detecting viral nucleic acids. The main problem with RT-PCR test is the false negative results. The negative RT-PCR does not exclude a SARS-CoV-2 infection and this method should not be used as the only diagnostic criteria. The RT-PCR result does not change the complex treatment of the disease. The aim of the current study is to compare the four groups clinical cases of the different parameters: RT-PCR test, rapid test, clinical picture, laboratory tests as hematology, inflammatory markers, coagulation status and chemistry and imaging examinations: Chest X-ray at and Chest CT scan. Complex therapeutic approach has been implemented: antibiotic, inflammatory, anticoagulants, oxygen therapy, hepatoprotectors, antimycotics, fibrinolytics, probiotics, essential oils, vitamins. During the follow-up period, a tendency for significant reduction and resorption of the pulmonary changes on the CT scans has been seen.

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