scholarly journals Inhibition monitoring in veterinary molecular testing

2019 ◽  
Vol 32 (6) ◽  
pp. 758-766 ◽  
Author(s):  
Lifang Yan ◽  
Kathy L. Toohey-Kurth ◽  
Beate M. Crossley ◽  
Jianfa Bai ◽  
Amy L. Glaser ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Internal Control ◽  
Control Strategies ◽  
False Negative ◽  
Internal Controls ◽  
Molecular Testing ◽  
Acid Extraction ◽  
Negative Results ◽  
False Negative Results ◽  
Laboratory Technology

Many of the sample matrices typically used for veterinary molecular testing contain inhibitory factors that can potentially reduce analytic sensitivity or produce false-negative results by masking the signal produced by the nucleic acid target. Inclusion of internal controls in PCR-based assays is a valuable strategy not only for monitoring for PCR inhibitors, but also for monitoring nucleic acid extraction efficiency, and for identifying technology errors that may interfere with the ability of an assay to detect the intended target. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians reviewed the different types of internal controls related to monitoring inhibition of PCR-based assays, and provides information here to encourage veterinary diagnostic laboratories to incorporate PCR internal control strategies as a routine quality management component of their molecular testing.

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 Evaluation of Three Automated Nucleic Acid Extraction Systems for Identification of Respiratory Viruses in Clinical Specimens by Multiplex Real-Time PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Yoonjung Kim ◽  
Mi-Soon Han ◽  
Juwon Kim ◽  
Aerin Kwon ◽  
Kyung-A Lee
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
False Negative ◽  
Virus Detection ◽  
Turnaround Time ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Nucleic Acid Extraction ◽  
Negative Results

A total of 84 nasopharyngeal swab specimens were collected from 84 patients. Viral nucleic acid was extracted by three automated extraction systems: QIAcube (Qiagen, Germany), EZ1 Advanced XL (Qiagen), and MICROLAB Nimbus IVD (Hamilton, USA). Fourteen RNA viruses and two DNA viruses were detected using the Anyplex II RV16 Detection kit (Seegene, Republic of Korea). The EZ1 Advanced XL system demonstrated the best analytical sensitivity for all the three viral strains. The nucleic acids extracted by EZ1 Advanced XL showed higher positive rates for virus detection than the others. Meanwhile, the MICROLAB Nimbus IVD system was comprised of fully automated steps from nucleic extraction to PCR setup function that could reduce human errors. For the nucleic acids recovered from nasopharyngeal swab specimens, the QIAcube system showed the fewest false negative results and the best concordance rate, and it may be more suitable for detecting various viruses including RNA and DNA virus strains. Each system showed different sensitivity and specificity for detection of certain viral pathogens and demonstrated different characteristics such as turnaround time and sample capacity. Therefore, these factors should be considered when new nucleic acid extraction systems are introduced to the laboratory.

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 Analysis and Prediction of COVID-19 Patients’ False Negative Results for SARS-CoV-2 Detection with Pharyngeal Swab Specimen: A Retrospective Study

2020 ◽  
Author(s):  
Hui Xu ◽  
Li Yan ◽  
Chun (Martin) Qiu ◽  
Bo Jiao ◽  
Yanyan Chen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Time Window ◽  
Clinical Symptoms ◽  
False Negative ◽  
Ct Imaging ◽  
Nucleic Acid Detection ◽  
False Negatives ◽  
Negative Results ◽  
False Negative Results ◽  
Blood Routine

ABSTRACTBackgroundFalse negative results of SARS-CoV-2 nucleic acid detection pose threats to COVID-19 patients and medical workers alike.ObjectiveTo develop multivariate models to determine clinical characteristics that contribute to false negative results of SARS-CoV-2 nucleic acid detection, and use them to predict false negative results as well as time windows for testing positive.DesignRetrospective Cohort Study (Ethics number of Tongji Hospital: No. IRBID: TJ-20200320)SettingA database of outpatients in Tongji Hospital (University Hospital) from 15 January 2020 to 19 February 2020.Patients1,324 outpatients with COVID-19MeasurementsClinical information on CT imaging reports, blood routine tests, and clinic symptoms were collected. A multivariate logistic regression was used to explain and predict false negative testing results of SARS-CoV-2 detection. A multivariate accelerated failure model was used to analyze and predict delayed time windows for testing positive.ResultsOf the 1,324 outpatients who diagnosed of COVID-19, 633 patients tested positive in their first SARS-CoV-2 nucleic acid test (47.8%), with a mean age of 51 years (SD=14.9); the rest, which had a mean age of 47 years (SD=15.4), tested negative in the first test. “Ground glass opacity” in a CT imaging report was associated with a lower chance of false negatives (aOR, 0.56), and reduced the length of time window for testing positive by 26%. “Consolidation” was associated with a higher chance of false negatives (aOR, 1.57), and extended the length of time window for testing positive by 44%. In blood routine tests, basophils (aOR, 1.28) and eosinophils (aOR, 1.29) were associated with a higher chance of false negatives, and were found to extend the time window for testing positive by 23% and 41%, respectively. Age and gender also affected the significantly.LimitationData were generated in a large single-center study.ConclusionTesting outcome and positive window of SARS-CoV-2 detection for COVID-19 patients were associated with CT imaging results, blood routine tests, and clinical symptoms. Taking into account relevant information in CT imaging reports, blood routine tests, and clinical symptoms helped reduce a false negative testing outcome. The predictive AFT model, what we believe to be one of the first statistical models for predicting time window of SARS-CoV-2 detection, could help clinicians improve the accuracy and efficiency of the diagnosis, and hence, optimizes the timing of nucleic acid detection and alleviates the shortage of nucleic acid detection kits around the world.Primary Funding SourceNone.

scholarly journals Development and application of a canine endogenous internal positive control for use in real-time PCR assays

2018 ◽  
Vol 30 (5) ◽  
pp. 789-792 ◽  
Author(s):  
Joseph J. Modarelli ◽  
Pamela J. Ferro ◽  
Maria D. Esteve-Gasent
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Positive Control ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Negative Results ◽  
False Negative Results ◽  
Internal Positive Control ◽  
Pcr Assays

Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats.

scholarly journals Prolonged nucleic acid conversion and false-negative RT-PCR results in Indonesian patients with COVID-19: A case series

2020 ◽  
Author(s):  
Ika Trisnawati ◽  
Riat Al Khair ◽  
Aditya Rifqi Fauzi ◽  
Gunadi
Keyword(s):  
Nucleic Acid ◽  
Male Patient ◽  
False Negative ◽  
Case Series ◽  
Heart Rhythm ◽  
X Rays ◽  
Rt Pcr ◽  
Negative Results ◽  
False Negative Results ◽  
Heart Rhythm Disorders

Abstract Background: Prolonged nucleic acid conversion and false-negative results of real-time reverse transcription polymerase chain reaction (RT-PCR) might occur in some patients with COVID-19 rather than recurrence of infection. Here, we reported four cases of COVID-19 with prolonged nucleic acid conversion and false-negative results of RT-PCR in our institution.Case presentation: Case 1: A 36-year-old-male patient complained of coughing up phlegm one week before admission. His chest X-rays showed mild pneumonia in the right lung. His swab test was confirmed positive for SARS-Cov-2. Besides the last two consecutive negative results, he also had negative results of RT-PCR twice (the 6th and 8th tests) from a total of 11 swab tests. Case 2: A 54-year-old-male patient complained of shortness of breath that worsened with activity. He had a comorbidity of diabetes. His chest X-rays showed inhomogeneous opacity on bilateral paracardial and lateral aspects. His swab test was confirmed positive for SARS-Cov-2. Besides the last two consecutive negative results, he also had negative results of RT-PCR once (the 5th test) from a total of 8 swab tests. Case 3: A 47-year-old man presented with complaints of fever, cough, sore throat, and diarrhea. He had comorbidities of asthma and heart rhythm disorders. His chest X-rays showed bilateral pneumonia. His swab test was confirmed positive for SARS-Cov-2. Besides the last two consecutive negative results, he also showed negative results of RT-PCR twice (the 4th and 6th tests) from a total of 11 swab tests. Case 4: A 56-year-old-female complained of lethargy and diarrhea. She has a history of hyperthyroidism. His chest X-rays showed bilateral pneumonia. She was confirmed positive for SARS-Cov-2. Besides the last two consecutive negative results, she also had negative results of RT-PCR twice (the 2nd and 10th tests) from a total of 14 swab tests.Conclusions: Our cases further confirmed the occurrence of prolonged nucleic acid conversion and the possibility of false negative results of RT-PCR in patients with COVID-19 instead of recurrence of infection. These findings might have an implication on the management of patients with COVID-19 who have already clinically and radiologically recovered, particularly related to subsequent spreading of the infection in the community.

scholarly journals Clinical Performance of SARS-CoV-2 Molecular Testing

2020 ◽  
Author(s):  
Daniel A. Green ◽  
Jason Zucker ◽  
Lars F. Westblade ◽  
Susan Whittier ◽  
Hanna Rennert ◽  
...  
Keyword(s):  
Clinical Performance ◽  
Severe Disease ◽  
False Negative ◽  
Molecular Testing ◽  
Repeat Testing ◽  
Clinical Sensitivity ◽  
Initial Testing ◽  
Molecular Assays ◽  
Negative Results ◽  
False Negative Results

AbstractMolecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the gold standard for diagnosis of coronavirus disease 2019 (COVID-19), but the test clinical performance is poorly understood. From 3/10/2020-5/1/2020 NewYork-Presbyterian laboratories performed 27,377 SARS-CoV-2 molecular assays from 22,338 patients. Repeat testing was performed in 3,432 patients, of which 2,413 had negative and 1,019 had positive first day results. Repeat-tested patients were more likely to be older, male, African-American or Hispanic, and to have severe disease. Among the patients with initially negative results, 18.6% became positive upon repeat-testing. Only 58.1% of any-time positive patients had a result of “detected” on the first test. The clinical sensitivity of COVID-19 molecular assays is estimated between 66.2 % and 95.6%, depending on the unknown number of false negative results in single-tested patients. Conversion to a negative result is unlikely to occur before 15 to 20 days after initial testing or 20-30 days after the onset of symptoms, with 50% conversion occurring at 28 days after initial testing. Forty-nine initially-positive patients converted to negative and then back to positive in subsequent days. Conversion from first day negative to positive results increased linearly with each day of testing, reaching 25% probability in 20 days. In summary, our study provides estimates of the clinical performance of SARS-CoV-2 molecular assays and suggests time frames for appropriate repeat testing, namely 15 to 20 days after a positive test and the same or next 2 days after a negative test in a patient with high suspicion for COVID-19.

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.

Evaluating real-time PCR for the quantification of distinct pathogens and indicator organisms in environmental samples

2004 ◽  
Vol 50 (1) ◽  
pp. 263-270 ◽  
Author(s):  
M. Lebuhn ◽  
M. Effenberger ◽  
G. Garcés ◽  
A. Gronauer ◽  
P.A. Wilderer
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
False Negative ◽  
Indicator Organisms ◽  
Negative Results ◽  
False Negative Results ◽  
Methodological Bias ◽  
Reliable Quantification

We evaluated quantitative real-time PCR (qPCR) and RTqPCR (for RNA species) for their ability to quantify microorganisms and viruses in problematic environmental samples such as cattle manure, digester material, wastewater and soil. Important developments included a standard spiking approach which compensated for methodological bias and allowed sample-to-sample comparison and reliable quantification. Programme CeTe was developed to calculate endogenous concentrations of target organisms (nucleic acid copies) for each sample separately from the generated standard curves. The approach also permitted assessment of the detection limit of the complete method, including extraction. It varied from sample to sample, due to different extraction efficiencies and variable co-extraction of PCR inhibitors. False negative results were thereby avoided. By using this approach we were able to optimise a DNA extraction protocol from the different tested sample types. Protocols for the extraction of RNA species from environmental samples were also optimised. DNA was (almost) not degraded after lethal shock (autoclaving) in the sterile environment. In contrast, the parallel selective cultivation and qPCR results for various microbial parameters from an anaerobic digester chain suggested that DNA from decaying organisms was readily recycled in metabolically active environments. It may, therefore, be used to determine viable organisms in samples exhibiting substantial metabolic turnover. It is proposed that our standard spiking approach, including data evaluation by the program CeTe, should be considered in future standardisation and norms for the quantification of nucleic acid containing organisms in environmental and product samples.

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