False negative results of real time strain elastography in thyroid nodular disease

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

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False Negative Results in High Viremia Parvovirus B19-Samples Tested with Real-Time PCR

Polish Journal of Microbiology ◽

10.33073/pjm-2010-020 ◽

2010 ◽

Vol 59 (2) ◽

pp. 129-132 ◽

Cited By ~ 4

Author(s):

PIOTR GRABARCZYK ◽

ALEKSANDRA KALIŃSKA ◽

EWA SULKOWSKA ◽

EWA BROJER

Keyword(s):

Positive Result ◽

Real Time ◽

Real Time Pcr ◽

Parvovirus B19 ◽

False Negative ◽

Immunocompromised Patients ◽

Negative Results ◽

False Negative Results ◽

Parvovirus B 19 ◽

The Way

Extremely high viremia is observed during some viruses infection, especialy in immunocompromised patients. False negative results of Parvovirus B 19 DNA tests performed with real-time PCR in high viremic samples are reported. The way of fluorescence diagrams analysis and algorithm of positive result confirmation to exclude such phenomenon are proposed.

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Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638717695609 ◽

2017 ◽

Vol 29 (3) ◽

pp. 351-356

Author(s):

Amaresh Das ◽

Ming Y. Deng ◽

Shawn Babiuk ◽

Michael T. McIntosh

Keyword(s):

Real Time ◽

False Negative ◽

Positive Control ◽

Lumpy Skin Disease ◽

Outbreak Management ◽

Negative Results ◽

Beta Actin ◽

False Negative Results ◽

Internal Positive Control ◽

Analytical Sensitivities

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

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SYBR Green-Based Real-Time Quantitative PCR Assay for Detection of West Nile Virus Circumvents False-Negative Results Due to Strain Variability

Journal of Clinical Microbiology ◽

10.1128/jcm.42.4.1511-1518.2004 ◽

2004 ◽

Vol 42 (4) ◽

pp. 1511-1518 ◽

Cited By ~ 85

Author(s):

J. F. Papin ◽

W. Vahrson ◽

D. P. Dittmer

Keyword(s):

West Nile Virus ◽

Real Time ◽

Quantitative Pcr ◽

False Negative ◽

Pcr Assay ◽

Real Time Quantitative Pcr ◽

Negative Results ◽

False Negative Results ◽

Strain Variability ◽

Quantitative Pcr Assay

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False-Negative Results of a Validated Real-Time PCR Protocol for Diagnosis of Newcastle Disease Due to Genetic Variability of the Matrix Gene

Journal of Clinical Microbiology ◽

10.1128/jcm.00895-09 ◽

2009 ◽

Vol 47 (11) ◽

pp. 3791-3792 ◽

Cited By ~ 24

Author(s):

G. Cattoli ◽

C. De Battisti ◽

S. Marciano ◽

S. Ormelli ◽

I. Monne ◽

...

Keyword(s):

Genetic Variability ◽

Real Time ◽

Newcastle Disease ◽

Real Time Pcr ◽

False Negative ◽

Negative Results ◽

Matrix Gene ◽

False Negative Results ◽

The Matrix

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Reducing False Negative PCR Test for COVID-19

International Journal of MCH and AIDS (IJMA) ◽

10.21106/ijma.421 ◽

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.

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Assessment of Commercial DNA Cleanup Kits for Elimination of Real-Time PCR Inhibitors in the Detection of Cyclospora cayetanensis in Cilantro

Journal of Food Protection ◽

10.4315/jfp-20-139 ◽

2020 ◽

Vol 83 (11) ◽

pp. 1863-1870

Author(s):

ANGELA ASSURIAN ◽

HELEN MURPHY ◽

ALICIA SHIPLEY ◽

HEDIYE NESE CINAR ◽

ALEXANDRE DA SILVA ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Threshold Values ◽

Cyclospora Cayetanensis ◽

Cycle Threshold ◽

Negative Results ◽

False Negative Results ◽

Pcr Inhibitor ◽

Zymo Research

ABSTRACT Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration–validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method. HIGHLIGHTS

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Contrast of Real-Time Fluorescent PCR Methods for Detection of Escherichia coli O157:H7 and of Introducing an Internal Amplification Control

Microorganisms ◽

10.3390/microorganisms7080230 ◽

2019 ◽

Vol 7 (8) ◽

pp. 230 ◽

Cited By ~ 3

Author(s):

Zhao ◽

Xia ◽

Liu

Keyword(s):

Real Time ◽

Water Samples ◽

False Negative ◽

Internal Amplification Control ◽

Pcr Assay ◽

The Real ◽

E Coli ◽

Negative Results ◽

False Negative Results ◽

Fluorescent Pcr

Various constituents in food specimens can inhibit the PCR assay and lead to false-negative results. An internal amplification control was employed to monitor the presence of false-negative results in PCR amplification. In this study, the objectives were to compare the real-time PCR-based method by introducing a competitive internal amplification control (IAC) for the detection of Escherichia O157:H7 with respect to the specificity of the primers and probes, analytical sensitivity, and detection limits of contamination-simulated drinking water. Additionally, we optimized the real-time fluorescent PCR detection system for E. coli O157:H7. The specificity of primers and probes designed for the rfbE gene was evaluated using four kinds of bacterial strains, including E. coli O157:H7, Staphylococcus aureus, Salmonella and Listeria monocytogenes strains. The real time PCR assay unambiguously distinguished the E. coli O157:H7 strains after 16 cycles. Simultaneously, the lowest detection limit for E. coli O157:H7 in water samples introducing the IAC was 104 CFU/mL. The analytical sensitivity in water samples had no influence on the detection limit compared with that of pure cultures. The inclusion of an internal amplification control in the real-time PCR assay presented a positive IAC amplification signal in artificially simulated water samples. These results indicated that real-time fluorescent PCR combined with the IAC possessed good characteristics of stability, sensitivity, and specificity. Consequently, the adjusted methods have the potential to support the fast and sensitive detection of E. coli O157:H7, enabling accurate quantification and preventing false negative results in E. coli O157:H7 contaminated samples.

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A Multiplex Real-Time Polymerase Chain Reaction (TaqMan) Assay for the Simultaneous Detection of Meloidogyne chitwoodi and M. fallax

Phytopathology ◽

10.1094/phyto-96-1255 ◽

2006 ◽

Vol 96 (11) ◽

pp. 1255-1262 ◽

Cited By ~ 32

Author(s):

C. Zijlstra ◽

R. A. Van Hoof

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Simultaneous Detection ◽

Chain Reaction ◽

Meloidogyne Chitwoodi ◽

Negative Results ◽

False Negative Results ◽

Polymerase Chain

This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-labeled to detect M. fallax, and one NED-labeled to detect the internal amplification control (IAC) to monitor false negative results. One common primer set is used for the amplification of part of the internal transcribed spacer (ITS) region of M. chitwoodi and M. fallax and one primer set for the amplification of the IAC. The test enabled detection of M. chitwoodi and/or M. fallax in DNA samples extracted from batches of juveniles, from single juveniles, and from infected plant material. Compared with current assays to detect M. chitwoodi and M. fallax, the multiplex real-time PCR offers the following advantages: it is faster because the test can simultaneously detect both quarantine species without the need for post-PCR processing; and it is at least 10 times more sensitive than a comparable regular PCR also targeting the ITS sequence. Inclusion of the IAC facilitates the interpretation of the FAM and VIC cycle threshold (Ct) values and can prevent the scoring of false negative results when FAM, VIC, and NED Ct values are high. The test allows precise quantification when only one of the two species is present in the sample. However, experiments with mixtures of genomic DNA of M. chitwoodi and M. fallax revealed that the ability of the multiplex real-time PCR assay to detect small quantities of DNA of one species is reduced when large quantities of DNA of the other species are present.

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