scholarly journals A SARS-CoV-2 Delta Variant Containing Mutation in the Probe Binding Region Used for qRT-PCR Test in Japan Exhibited Atypical PCR Amplification and Might Induce False Negative Result

2021 ◽  
Author(s):  
Samiul Alam Rajib ◽  
Yasuhiro Ogi ◽  
Md Belal Hossain ◽  
Terumasa Ikeda ◽  
Eiichi Tanaka ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Genome Sequencing ◽  
De Novo ◽  
False Negative ◽  
Pcr Amplification ◽  
Coding Region ◽  
Economic Activities ◽  
Qrt Pcr ◽  
Negative Results ◽  
Nucleotide Mutation

A recent pandemic of SARS-CoV-2 infection has caused severe health problems and substantially restricted social and economic activities. To cope with such an outbreak, the identification of infected individuals with high accuracy is vital. qRT-PCR plays a key role in the diagnosis of SARS-CoV-2 infection. The N protein-coding region is widely analyzed in qRT-PCR for the diagnosis of SARS-CoV-2 infection in Japan. We recently encountered two cases of SARS-CoV-2-positive specimens showing atypical amplification curves in the qRT-PCR. We performed whole-genome sequencing and found that the virus was a Delta-type variant of SARS-CoV-2 with a single nucleotide mutation in the probe-binding site. To evaluate the extent of spread of the variant in the area, we performed whole viral genome sequencing of samples collected from 61 patients infected with SARS-CoV-2 during the same time and in the same area. There were no other cases with the same mutation, indicating that the variant had not spread in the area. Furthermore, we performed phylogenetic analysis with various SARS-CoV-2 sequences deposited in the public database. Hundreds of variants were reported globally, and one in Japan were found to contain the same mutation. Phylogenetic analysis showed that the variant was very close to other Delta variants endemic in Japan but quite far from the variants containing the same mutation reported from outside Japan, suggesting that the variant would have been sporadically generated in some domestic areas. These findings propose two key points: i) mutations in the region used for SARS-CoV-2 qRT-PCR can cause abnormal amplification curves; therefore, the qRT-PCR result should not just be judged in an automated manner, but also manually checked by the examiner to prevent false-negative results, and ii) various mutations can be generated sporadically and unpredictably; therefore, efficient and robust screening systems are needed to promptly monitor the emergence of de novo variants.

scholarly journals Emerging approaches for detection of methylation sites in RNA

Open Biology ◽  
2018 ◽  
Vol 8 (9) ◽  
pp. 180121 ◽  
Author(s):  
Anna Ovcharenko ◽  
Andrea Rentmeister
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Direct Detection ◽  
False Negative ◽  
Detection Methods ◽  
Rna Modifications ◽  
Negative Results ◽  
Third Generation Sequencing ◽  
False Negative Results ◽  
Generation Sequencing

RNA methylations play a significant regulatory role in diverse biological processes. Although the transcriptome-wide discovery of unknown RNA methylation sites is essential to elucidate their function, the development of a bigger variety of detection approaches is desirable for multiple reasons. Many established detection methods for RNA modifications heavily rely on the specificity of the respective antibodies. Thus, the development of antibody-independent transcriptome-wide methods is beneficial. Even the antibody-independent high-throughput sequencing-based methods are liable to produce false-positive or false-negative results. The development of an independent method for each modification could help validate the detected modification sites. Apart from the transcriptome-wide methods for methylation detection de novo , methods for monitoring the presence of a single methylation at a determined site are also needed. In contrast to the transcriptome-wide detection methods, the techniques used for monitoring purposes need to be cheap, fast and easy to perform. This review considers modern approaches for site-specific detection of methylated nucleotides in RNA. We also discuss the potential of third-generation sequencing methods for direct detection of RNA methylations.

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 Mutation Detection in the Menkes Gene ATP7A Using the Protein Truncation Test

2008 ◽  
Vol 1 ◽  
pp. CPath.S565 ◽  
Author(s):  
Lisbeth Birk Møller ◽  
Nina Horn
Keyword(s):  
False Negative ◽  
Copper Metabolism ◽  
Menkes Disease ◽  
Allelic Exclusion ◽  
Missense Mutations ◽  
Coding Region ◽  
Protein Truncation Test ◽  
Exon 2 ◽  
Negative Results ◽  
Protein Truncation

Menkes disease (MD) is a rare recessively inherited lethal disorder of copper metabolism. The gene ATP7A defective in MD consists of 23 exons and the coding region encompasses 4500 bp. About 300 distinct mutations, representing all types, have been identified in ATP7A. However all mutations identified so far in the exon 2 to exon 7, corresponding to 1869 bp of the coding sequence, result in truncated protein products. No missense mutations have been identified in this region. As about 30% of the total number of mutations identified are located in exon 2 to exon 7, we have designed a protein truncation test (PTT) for rapid detecting of mutations in this part of the gene. In order to determine the applicability of the test, we analysed RNA obtained from eleven MD patients with known mutations in this region. As a truncated product could be identified in all the included samples, PTT proves to be a useful technique for rapid detection of mutations in the N-terminal part of the ATP7A gene. Furthermore as MD is a X-linked disease, normally only affecting boys, the risk of false negative results, due to nonsense mediated RNA decay, leading to allelic exclusion, can be left out of account.

scholarly journals Analysis of Lewis fucosyltransferase genes from the human gastric mucosa of Lewis-positive and -negative individuals

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2915-2919 ◽  
Author(s):  
Y Koda ◽  
H Kimura ◽  
E Mekada
Keyword(s):  
Gastric Mucosa ◽  
Pcr Amplification ◽  
Base Change ◽  
Human Gastric Mucosa ◽  
Coding Region ◽  
Single Base ◽  
Cos Cells ◽  
Lewis Antigen ◽  
Base Substitutions ◽  
Nucleotide Mutation

Abstract The expression of Lewis fucosyltransferase (FT) mRNA was examined in gastric mucosa from two Lewis-positive [Le(+)] and two Lewis-negative [Le(-)] individuals. Northern blot analysis demonstrated that levels of mRNA were similar in both Le(+) and Le(-) gastric mucosa. We isolated the protein-coding region of the Lewis FT cDNA from Le(+) and Le(-) gastric mucosa by polymerase chain reaction (PCR) amplification. The sequence of cDNA from the Le(-) gastric mucosa shows two single-base substitutions of G for T at position 59 and of A for G at position 508 from the A of the initiation codon of cDNA. These substitutions may be the cause of changes in two amino acid residues, Arg for Leu at position 20 and Ser for Gly at position 170 from the N-terminal. To determine whether either or both of these base substitutions is responsible for the Le(-) gene, we constructed chimera cDNAs and expressed them in COS cells. Those COS cells transfected with a chimera cDNA containing a mutation of the 508th nucleotide did not express Lewis antigen, whereas those cells transfected with a chimera cDNA containing the 59th nucleotide mutation expressed Lewis antigen, indicating that a single-base change from G to A at position 508 is responsible for the Le(-) gene. The G to A transition at position 508 created a new site for PvuII endonuclease. The digestion by PvuII endonuclease of PCR products between the 386th and 612th nucleotides of Lewis FT cDNA from one of the Le(-) individuals proved to be homozygous for the PvuII site. However, the other Le(-) individual was heterozygous for the PvuII site, suggesting the presence of other Le(-) allele(s). Thus, we isolated one of the silent Lewis genes (le).

scholarly journals Analysis of Lewis fucosyltransferase genes from the human gastric mucosa of Lewis-positive and -negative individuals

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2915-2919 ◽  
Author(s):  
Y Koda ◽  
H Kimura ◽  
E Mekada
Keyword(s):  
Gastric Mucosa ◽  
Pcr Amplification ◽  
Base Change ◽  
Human Gastric Mucosa ◽  
Coding Region ◽  
Single Base ◽  
Cos Cells ◽  
Lewis Antigen ◽  
Base Substitutions ◽  
Nucleotide Mutation

The expression of Lewis fucosyltransferase (FT) mRNA was examined in gastric mucosa from two Lewis-positive [Le(+)] and two Lewis-negative [Le(-)] individuals. Northern blot analysis demonstrated that levels of mRNA were similar in both Le(+) and Le(-) gastric mucosa. We isolated the protein-coding region of the Lewis FT cDNA from Le(+) and Le(-) gastric mucosa by polymerase chain reaction (PCR) amplification. The sequence of cDNA from the Le(-) gastric mucosa shows two single-base substitutions of G for T at position 59 and of A for G at position 508 from the A of the initiation codon of cDNA. These substitutions may be the cause of changes in two amino acid residues, Arg for Leu at position 20 and Ser for Gly at position 170 from the N-terminal. To determine whether either or both of these base substitutions is responsible for the Le(-) gene, we constructed chimera cDNAs and expressed them in COS cells. Those COS cells transfected with a chimera cDNA containing a mutation of the 508th nucleotide did not express Lewis antigen, whereas those cells transfected with a chimera cDNA containing the 59th nucleotide mutation expressed Lewis antigen, indicating that a single-base change from G to A at position 508 is responsible for the Le(-) gene. The G to A transition at position 508 created a new site for PvuII endonuclease. The digestion by PvuII endonuclease of PCR products between the 386th and 612th nucleotides of Lewis FT cDNA from one of the Le(-) individuals proved to be homozygous for the PvuII site. However, the other Le(-) individual was heterozygous for the PvuII site, suggesting the presence of other Le(-) allele(s). Thus, we isolated one of the silent Lewis genes (le).

scholarly journals PCR Primer Specific CaMV 35S Promoter to Detect Transgenic Soybean in Indonesia Commercial Soy Bean and Tempeh

2017 ◽  
Vol 17 (3) ◽  
pp. 415 ◽  
Author(s):  
Tri Joko Raharjo ◽  
Surajiman Surajiman
Keyword(s):  
False Negative ◽  
Pcr Amplification ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Good Precision ◽  
Transgenic Soybean ◽  
Camv 35S ◽  
Negative Results ◽  
False Negative Results ◽  
Total Dna

In the framework of the supervision and enforcement of the regulation regarding the content of soybean transgenic in food and processed foods such as tempeh, a reliable testing method is indispensable. Performance specific primer PCR amplification with promoter of CaMV 35S tested to detect the presence of GMOs. The parameters tested were specificity, precision and cut off detection using CRM transgenic soybean. The method is reliable to detect transgenic soybean specifically and has the annealing temperature at 59 °C during the 30 cycle standard PCR condition. The method did not show any false positive and false negative results meaning good precision. The cut off the methods is up to 2 copies total DNA of soybean or less than 104 copies of the CaMV 35S promoter. Observation to the commercial soybeans and tempeh found that most of the commercially available soybean in Indonesia are transgenic (8 of 10 sample) while all tested tempeh sample were detected have been fermented from transgenic soybeans.

scholarly journals Interpretation of Negative Molecular Test Results in Patients With Suspected or Confirmed Ebola Virus Disease: Report of Two Cases

2015 ◽  
Vol 2 (4) ◽  
Author(s):  
Jeffrey K. Edwards ◽  
Christian Kleine ◽  
Vincent Munster ◽  
Ruggero Giuliani ◽  
Moses Massaquoi ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Virus Disease ◽  
False Negative ◽  
Ebola Virus Disease ◽  
Test Results ◽  
Qrt Pcr ◽  
Negative Results ◽  
First Case ◽  
False Negative Results ◽  
Late Stages

Abstract Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is the most sensitive quantitative diagnostic assay for detection of Ebola virus in multiple body fluids. Despite the strengths of this assay, we present 2 cases of Ebola virus disease (EVD) and highlight the potential for false-negative results during the early and late stages of EVD. The first case emphasizes the low negative-predictive value of qRT-PCR during incubation and the early febrile stage of EVD, and the second case emphasizes the potential for false-negative results during recovery and late neurologic complications of EVD. Careful interpretation of test results are needed to guide difficult admission and discharge decisions in suspected or confirmed EVD.

scholarly journals Application of Whole-Genome Sequencing for Bacterial Strain Typing in Molecular Epidemiology

2015 ◽  
Vol 53 (4) ◽  
pp. 1072-1079 ◽  
Author(s):  
Stephen J. Salipante ◽  
Dhruba J. SenGupta ◽  
Lisa A. Cummings ◽  
Tyler A. Land ◽  
Daniel R. Hoogestraat ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Gold Standard ◽  
False Negative ◽  
Laboratory Method ◽  
Whole Genome ◽  
Strain Typing ◽  
Content Type ◽  
Negative Results ◽  
Quantitative Definition

Nosocomial infections pose a significant threat to patient health; however, the gold standard laboratory method for determining bacterial relatedness (pulsed-field gel electrophoresis [PFGE]) remains essentially unchanged 20 years after its introduction. Here, we explored bacterial whole-genome sequencing (WGS) as an alternative approach for molecular strain typing. We compared WGS to PFGE for investigating presumptive outbreaks involving three important pathogens: vancomycin-resistantEnterococcus faecium(n= 19), methicillin-resistantStaphylococcus aureus(n= 17), andAcinetobacter baumannii(n= 15). WGS was highly reproducible (average ≤ 0.39 differences between technical replicates), which enabled a functional, quantitative definition for determining clonality. Strain relatedness data determined by PFGE and WGS roughly correlated, but the resolution of WGS was superior (P= 5.6 × 10−8to 0.016). Several discordant results were noted between the methods. A total of 28.9% of isolates which were indistinguishable by PFGE were nonclonal by WGS. ForA. baumannii, a species known to undergo rapid horizontal gene transfer, 16.2% of isolate pairs considered nonidentical by PFGE were clonal by WGS. Sequencing whole bacterial genomes with single-nucleotide resolution demonstrates that PFGE is prone to false-positive and false-negative results and suggests the need for a new gold standard approach for molecular epidemiological strain typing.

scholarly journals Establishment of Simultaneous Detection and Quantitation of HCVRNA by Third Generation Intercalating Dye Real-time PCR

2018 ◽  
Vol 17 (1) ◽  
Author(s):  
Akrahm M. Saleh Habil ◽  
Hairul Aini Hamzah ◽  
Muhammad Imad Al-Deen Mustafa ◽  
Norlelawati A. Talib ◽  
Siti Nurul Fazlin Abdul Rahman
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
False Negative ◽  
Simultaneous Detection ◽  
Pcr Amplification ◽  
Serum Samples ◽  
Negative Results ◽  
Broad Dynamic Range

Introduction: Rapid quantification of hepatitis C virus is helpful in determining and monitoring of the disease progression and nature of the virus replication. The aim of the present study was to establish a fast, specific and sensitive tool for HCVRNA quantification. Materials and Methods: A total of 50 serum samples, comprising of 40 HCV-positive and 10 HCV-negative, were included in our study. RNA was extracted, reverse transcribed, and then subjected to real-time PCR amplification. Real-time PCR using EvaGreen dye and primers targeting a 5’UTR was carried out. Reference samples with known viral load were treated similarly to the unknown samples and used to create the standard curves. Results: Our method showed a high level of analytical specificity and accuracy, with a low limit of detection (~2 IU/ml). It yielded repeatable results with less than 4% of intra- assay variation. The assay covered a broad dynamic range of quantification, ranging from 0.34 to 6 log IU/ml. The diagnostic sensitivity, specificity, and accuracy were all 100%, indicating neither false positive nor false negative results were obtained. Conclusion: The developed real time PCR using EvaGreen dye has demonstrated a highly analytical and diagnostic performance for HCV quantification, suggesting its potential in clinical diagnosis and management.

scholarly journals Identification and Characterization of a Cryptic Genomic Deletion-Insertion in EYA1 Associated with Branchio-Otic Syndrome

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Hao Zheng ◽  
Jun Xu ◽  
Yu Wang ◽  
Yun Lin ◽  
Qingqiang Hu ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Genome Sequencing ◽  
De Novo ◽  
Pcr Amplification ◽  
Genomic Alteration ◽  
Causative Gene ◽  
Genomic Deletion ◽  
Targeted Next Generation Sequencing ◽  
Ear Malformations ◽  
Multiple Regions

Branchio-oto-renal spectrum disorder (BORSD) is characterized by hearing loss accompanied by ear malformations, branchial cysts, and fistulae, with (branchio-oto-renal syndrome (BORS)) or without renal abnormalities (BOS (branchio-otic syndrome)). As the most common causative gene for BORSD, dominant mutations in EYA1 are responsible for approximately 40% of the cases. In a sporadic deaf patient diagnosed as BOS, we identified an apparent heterozygous genomic deletion spanning the first four coding exons and one 5 ′ noncoding exon of EYA1 by targeted next-generation sequencing of 406 known deafness genes. Real-time PCR at multiple regions of EYA1 confirmed the existence of this genomic deletion and extended its 5 ′ boundary beyond the 5 ′ -UTR. Whole genome sequencing subsequently located the 5 ′ and 3 ′ breakpoints to 19268 bp upstream to the ATG initiation codon and 3180 bp downstream to exon 5. PCR amplification across the breakpoints in both the patient and his parents showed that the genomic alteration occurred de novo. Sanger sequencing of this PCR product revealed that it is in fact a GRCh38/hg38:chr8:g.71318554_71374171delinsTGCC genomic deletion-insertion. Our results showed that the genomic variant is responsible for the hearing loss associated with BOS and provided an example for deciphering such cryptic genomic alterations following pipelines of comprehensive exome/genome sequencing and designed verification.

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