scholarly journals Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes

2021 ◽  
Author(s):  
Enos C Kline ◽  
Nuttada Panpradist ◽  
Ian T Hull ◽  
Qin Wang ◽  
Amy K Oreskovic ◽  
...  
Keyword(s):  
Molecular Diagnostics ◽  
Detection System ◽  
Point Of Care ◽  
False Negative ◽  
Cost Effective ◽  
Internal Amplification Control ◽  
Robust Detection ◽  
Negative Results ◽  
False Negative Results ◽  
Target Redundancy

AbstractThe increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multi-target redundancy and an internal amplification control. A convenient and cost-effective target specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-COV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multi-target redundancy. When challenged with extracted human samples the multiplexed assay showed 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity. These results are further evidence that conventional laboratory methodologies can be leveraged at the point-of-care for robust performance and diagnostic stability over time.

Development of a recombinase polymerase amplification assay forVibrio parahaemolyticusdetection with an internal amplification control

2018 ◽  
Vol 64 (4) ◽  
pp. 223-230 ◽  
Author(s):  
Huan-Lan Yang ◽  
Shuang Wei ◽  
Ravi Gooneratne ◽  
Anthony N. Mutukumira ◽  
Xue-Jun Ma ◽  
...  
Keyword(s):  
Bacterial Species ◽  
False Negative ◽  
Recombinase Polymerase Amplification ◽  
Internal Amplification Control ◽  
Target Sequence ◽  
Negative Results ◽  
False Negative Results ◽  
Amplification Assay ◽  
Pcr Method ◽  
Recombinase Polymerase Amplification Assay

A novel RPA–IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 103CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA–IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

scholarly journals Interference of Asfotase Alfa in Immunoassays Employing Alkaline Phosphatase Technology

2019 ◽  
Vol 5 (2) ◽  
pp. 290-299
Author(s):  
Isabelle Danielle Piec ◽  
Beatrice Tompkins ◽  
William Duncan Fraser
Keyword(s):  
Alkaline Phosphatase ◽  
False Positive ◽  
Troponin I ◽  
Detection System ◽  
False Negative ◽  
Detection Systems ◽  
Asfotase Alfa ◽  
Negative Results ◽  
False Negative Results ◽  
Alternative Detection

Abstract Background Asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.) is the only approved treatment for patients with pediatric-onset hypophosphatasia, a disease caused by a mutation in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. ALP is often used as signaling system in routine immunoassays. Because asfotase alfa contains the active site of the full ALP enzyme, it can catalyze the substrate as the antibody-conjugated ALP would within an assay. Therefore, its presence in a treated patient’s sample may generate false positive or false negative results. We investigated whether the presence of asfotase alfa within a sample induced interference in immunoassays that utilize ALP or alternative detection systems. Methods Asfotase alfa was added to samples at concentrations from 0.08–5 µg/mL and analysed on various immunoassays following manufacturer’s instructions. Results Asfotase alfa was detected in all ALP assays but ALKP1 (RayBiotech). We observed no changes in normetanephrine and noradrenaline (IBL) at any asfotase alfa concentration. However, asfotase alfa notably interfered in an oxytocin (ENZO) assay in nonextracted samples. Extraction using a C18 column eliminated the interference. No interference was observed on automated analyzers using alternative detection system (COBAS fT4 and TSH; Advia Centaur FSH, fT4; Architect LH; FSH). Immulite 2000 fT4, TSH, testosterone and hCG (ALP-based) showed no interference. However, the presence of asfotase alfa resulted in a dose-dependent increase of Troponin I signal. Conclusion The presence of asfotase alfa must be taken into consideration when analyzing blood samples in treated patients to avoid any risk of misinterpretation of false positive/negative results. It is essential that assays be tested for this possible interference.

scholarly journals False-Negative Results in Point-of-Care Qualitative Human Chorionic Gonadotropin (hCG) Devices Due to Excess hCGβ Core Fragment

2009 ◽  
Vol 55 (7) ◽  
pp. 1389-1394 ◽  
Author(s):  
Ann M Gronowski ◽  
Mark Cervinski ◽  
Ulf-Håkan Stenman ◽  
Alison Woodworth ◽  
Lori Ashby ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Point Of Care ◽  
False Negative ◽  
The Core ◽  
Core Fragment ◽  
Negative Results ◽  
False Negative Results ◽  
Positive Results ◽  
Urine Specimens

Abstract Background: During pregnancy, human chorionic gonadotropin (hCG) immunoreactivity in urine consists of intact hCG as well as a number of hCG variants including the core fragment of hCGβ (hCGβcf). We identified 3 urine specimens with apparent false-negative results using the OSOM® hCG Combo Test (Genzyme Diagnostics) qualitative hCG device and sought to determine whether an excess of 1 of the fragments or variants might be the cause of the interference. Methods: We measured concentrations of hCG variants in the urine from 3 patients with apparent false-negative hCG results. Purified hCG variants were added to urines positive for hCG and tested using the OSOM, ICON® 25 hCG (Beckman Coulter), and hCG Combo SP® Brand (Cardinal Health) devices. Results: Dilution of these 3 urine samples resulted in positive results on the OSOM device. Quantification of hCG variants in each of the 3 patient urine specimens demonstrated that hCGβcf occurred in molar excess of intact hCG. Addition of purified hCGβcf to hCG-positive urines caused false-negative hCG results using the OSOM and ICON qualitative urine hCG devices. Conclusions: Increased concentrations of hCGβcf can cause false-negative results on the OSOM and ICON qualitative urine hCG devices. .

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

2019 ◽  
Vol 7 (8) ◽  
pp. 230 ◽  
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.

scholarly journals IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2

2020 ◽  
Author(s):  
Elizabeth C. Stahl ◽  
Connor A. Tsuchida ◽  
Jennifer R. Hamilton ◽  
Enrique Lin-Shiao ◽  
Shana L. McDevitt ◽  
...  
Keyword(s):  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
N Gene ◽  
Negative Results ◽  
False Negative Results ◽  
Single Well ◽  
Sample Pooling ◽  
One Step ◽  
Viral Target

AbstractCommonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.Graphical Abstract

scholarly journals Performance of SARS-CoV-2 antigen testing in symptomatic and asymptomatic adults: a single-center evaluation

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Stephanie L. Mitchell ◽  
Steven Orris ◽  
Tanner Freeman ◽  
Megan C. Freeman ◽  
Michelle Adam ◽  
...  
Keyword(s):  
Symptom Onset ◽  
Point Of Care ◽  
False Negative ◽  
Poor Performance ◽  
Nasopharyngeal Swab ◽  
Lower Sensitivity ◽  
Negative Results ◽  
Asymptomatic Patients ◽  
False Negative Results ◽  
Antigen Testing

Abstract Background Antigen testing offers rapid and inexpensive testing for SARS-CoV-2 but concerns regarding performance, especially sensitivity, remain. Limited data exists for use of antigen testing in asymptomatic patients; thus, performance and reliability of antigen testing remains unclear. Methods 148 symptomatic and 144 asymptomatic adults were included. A nasal swab was collected for testing by Quidel Sofia SARS IFA (Sofia) as point of care. A nasopharyngeal swab was also collected and transported to the laboratory for testing by Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV RT-PCR (Cepheid). Results Overall, Sofia had good agreement with Cepheid (> 95%) in adults, however was less sensitive. Sofia had a sensitivity of 87.8% and 33.3% for symptomatic and asymptomatic patients, respectively. Among symptomatic patients, testing > 5 days post symptom onset resulted in lower sensitivity (82%) when compared with testing within 5 days of symptom onset (90%). Of the four Sofia false-negative results in the asymptomatic cohort, 50% went on to develop COVID-19 disease within 5 days of testing. Specificity in both symptomatic and asymptomatic cohorts was 100%. Conclusions Sofia has acceptable performance in symptomatic adults when tested < 5 days of symptom onset. Caution should be taken when testing patients with ≥ 5 days of symptoms. The combination of low prevalence and reduced sensitivity results in relatively poor performance of in asymptomatic patients. NAAT-based diagnostic assays should be considered in when antigen testing is unreliable, particularly in symptomatic patients with > 5 days of symptom onset and asymptomatic patients.

scholarly journals High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

2020 ◽  
Author(s):  
Sanchita Bhadra ◽  
Timothy E. Riedel ◽  
Simren Lakhotia ◽  
Nicholas D. Tran ◽  
Andrew D. Ellington
Keyword(s):  
Point Of Care ◽  
False Negative ◽  
Human Saliva ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Target Sequence ◽  
One Pot ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

ABSTRACTIsothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.

scholarly journals The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Soumitesh Chakravorty ◽  
Ann Marie Simmons ◽  
Mazhgan Rowneki ◽  
Heta Parmar ◽  
Yuan Cao ◽  
...  
Keyword(s):  
False Positive ◽  
Point Of Care ◽  
False Negative ◽  
World Health ◽  
Negative Results ◽  
Operational Characteristics ◽  
False Negative Results ◽  
Smear Negative ◽  
Rifampin Resistance ◽  
Health Organization

ABSTRACT The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection. IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R. IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R.

Sign in / Sign up

Export Citation Format

Share Document