scholarly journals Improved Real-Time PCR Diagnosis of Citrus Stubborn Disease by Targeting Prophage Genes of Spiroplasma citri

Plant Disease ◽  
2015 ◽  
Vol 99 (1) ◽  
pp. 149-154 ◽  
Author(s):  
Xuefeng Wang ◽  
Harsha Doddapaneni ◽  
Jianchi Chen ◽  
Raymond K. Yokomi
Keyword(s):  
False Negative ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Detection Sensitivity ◽  
Spiroplasma Citri ◽  
Negative Results ◽  
Enhanced Sensitivity ◽  
Field Samples ◽  
Primer Sets ◽  
Citrus Stubborn Disease

Spiroplasma citri is a phloem-limited bacterium causing citrus stubborn disease (CSD). Isolation and culturing of S. citri is technically demanding and time consuming. S. citri is typically low in titer and unevenly distributed in citrus, making reliable detection challenging. The current preferred detection method is polymerase chain reaction (PCR) assays with primers developed from sequences of S. citri housekeeping genes. Recent genome sequencing of S. citri revealed that the bacterium harbors multiple copies of prophage genes. Therefore, targeting multicopy prophage genes was hypothesized to improve sensitivity of PCR detection. Two primer sets, Php-orf1 and Php-orf3, were developed from conserved prophage sequences in the S. citri genome. These primer sets were used to evaluate detection sensitivity in SYBR Green-based quantitative PCR (qPCR) assays with 18 S. citri in cultures isolated from different hosts and locations. Prophage primer set Php-orf1 increased detection sensitivity by 4.91 and 3.65 cycle threshold (Cq) units compared with housekeeping gene primers for spiralin and P58 putative adhesin gene, respectively. Detection was slightly less sensitive for the Php-orf3 primer set at 3.02 and 1.76 Cq units, respectively, over the same housekeeping gene primers. The prophage primer sets were validated for qPCR detection with field samples from three citrus orchards in California's San Joaquin Valley collected from 2007 to 2013. The data showed that S. citri prophage sequences improved sensitivity for qPCR detection of S. citri-infected trees at least 10-fold and reduced the number of false-negative results. No false-positive samples were detected with any of the primer sets. The enhanced sensitivity resulted from the higher copy number of prophage genes in the S. citri genome and, thus, improved CSD diagnosis from field samples.

scholarly journals A Rapid and Low-Cost protocol for the detection of B.1.1.7 lineage of SARS-CoV-2 by using SYBR Green-Based RT-qPCR

2021 ◽  
Author(s):  
Fadi Abdel Sater ◽  
Mahmoud Younes ◽  
Hassan Nassar ◽  
Paul Nguewa ◽  
Kassem Hamze
Keyword(s):  
Low Cost ◽  
False Negative ◽  
Sybr Green ◽  
Reproductive Number ◽  
Rt Pcr ◽  
Negative Results ◽  
False Negative Results ◽  
New Variant ◽  
Primer Sets ◽  
The Uk

AbstractBackgroundThe new SARS-CoV-2 variant VUI (202012/01), identified recently in the United Kingdom (UK), exhibits a higher transmissibility rate compared to other variants, and a reproductive number 0.4 higher. In the UK, scientists were able to identify the increase of this new variant through the rise of false negative results for the spike (S) target using a three-target RT-PCR assay (TaqPath kit).MethodsTo control and study the current coronavirus pandemic, it is important to develop a rapid and low-cost molecular test to identify the aforementioned variant. In this work, we designed primer sets specific to SARS-CoV-2 variant VUI (202012/01) to be used by SYBR Green-based RT-PCR. These primers were specifically designed to confirm the deletion mutations Δ69/Δ70 in the spike and the Δ106/Δ107/Δ108 in the NSP6 gene. We studied 20 samples from positive patients, 16 samples displayed an S-negative profile (negative for S target and positive for N and ORF1ab targets) and four samples with S, N and ORF1ab positive profile.ResultsOur results emphasized that all S-negative samples harbored the mutations Δ69/Δ70 and Δ106/Δ107/Δ108. This protocol could be used as a second test to confirm the diagnosis in patients who were already positive to COVID-19 but showed false negative results for S-gene.ConclusionsThis technique may allow to identify patients carrying the VUI (202012/01) variant or a closely related variant, in case of shortage in sequencing.

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

Variables affecting laboratory diagnosis of acute rickettsial infection

2018 ◽  
Vol 39 (4) ◽  
pp. 220
Author(s):  
Cecilia Kato
Keyword(s):  
Molecular Detection ◽  
False Negative ◽  
Laboratory Diagnosis ◽  
Acute Stage ◽  
Detection Methods ◽  
Rickettsial Infection ◽  
Negative Results ◽  
Enhanced Sensitivity ◽  
False Negative Results ◽  
Antibody Titres

The reference standard for the confirmation of a recent rickettsial infection is by the observation of a four-fold or greater rise in antibody titres when testing paired acute and convalescent (two to four weeks after illness resolution) sera by serological assays (Figure 1). At the acute stage of illness, diagnosis is performed by molecular detection methods most effectively on DNA extracted from tissue biopsies (eschars, skin rash, and organs) or eschar swabs. Less invasive and more convenient samples such as blood and serum may also be used for detection; however, the low number of circulating bacteria raises the possibility of false negative results. Optimal sampling practices and enhanced sensitivity must therefore be considered in order to provide a more accurate laboratory diagnosis.

False-negative Chlamydia polymerase chain reaction result caused by a cryptic plasmid-deficient Chlamydia trachomatis strain in Australia

Sexual Health ◽  
2019 ◽  
Vol 16 (4) ◽  
pp. 394 ◽  
Author(s):  
Emma L. Sweeney ◽  
Cheryl Bletchly ◽  
Rita Gupta ◽  
David M. Whiley
Keyword(s):  
Polymerase Chain Reaction ◽  
Chlamydia Trachomatis ◽  
False Negative ◽  
Cryptic Plasmid ◽  
Chain Reaction ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain ◽  
Primer Sets ◽  
Pcr Targeting

Background The 7.5-kb chlamydial cryptic plasmid remains a widely used sequence target for Chlamydia trachomatis nucleic acid amplification tests, but sequence variation in this plasmid, particularly a previously reported 377-bp deletion, can cause false-negative results. Here we report the presence in Australia of a C. trachomatis strain lacking the cryptic plasmid. Methods: A rectal swab from a male in his 50s provided a positive result for C. trachomatis using the Roche Cobas 4800 test, but a negative result in our confirmatory in-house polymerase chain reaction (PCR) method targeting the chlamydial cryptic plasmid. This result was unexpected given our in-house PCR assay targeted a region of sequence outside the recognised 377-bp deletion. To further investigate this discrepancy, the sample was retested using a second in-house PCR targeting a chromosomal (ompA) gene as well as six primer sets flanking various regions of the cryptic plasmid. Results: The sample provided positive results in the second in-house method, confirming the presence of C. trachomatis DNA. All other primer sets targeting the cryptic plasmid failed to amplify, indicating a lack of the chlamydial cryptic plasmid in this sample. Conclusions: The recognition of a plasmid-deficient strain of C. trachomatis within Australia highlights further limitations of using the chlamydial cryptic plasmid for C. trachomatis diagnostics and re-emphasises the benefits of using multitarget assays to avoid false-negative results.

scholarly journals Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

2012 ◽  
Vol 54 (5) ◽  
pp. 245-248 ◽  
Author(s):  
Maria Cristina Carvalho do Espírito-Santo ◽  
Mónica Viviana Alvarado-Mora ◽  
Pedro Luiz Silva Pinto ◽  
Flair José Carrilho ◽  
João Renato Rebello Pinho ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
False Negative ◽  
Public Health Problem ◽  
Parasite Load ◽  
Detection Sensitivity ◽  
Major Public Health Problem ◽  
Guanidine Isothiocyanate ◽  
Negative Results ◽  
False Negative Results ◽  
Low Prevalence

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

scholarly journals Laboratory Diagnosis of African Horse Sickness: Comparison of Serological Techniques and Evaluation of Storage Methods of Samples for Virus Isolation

1990 ◽  
Vol 2 (1) ◽  
pp. 44-50 ◽  
Author(s):  
Carol House ◽  
Peter E. Mikiciuk ◽  
Mary Lou Beminger
Keyword(s):  
Virus Isolation ◽  
Fluorescent Antibody ◽  
False Negative ◽  
Laboratory Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
African Horse Sickness ◽  
Serum Samples ◽  
Negative Results ◽  
Field Samples ◽  
Storage Methods

Five serological methods of diagnosing African horse sickness were evaluated, using a battery of serum samples from experimental horses vaccinated and challenged with each serotype of African horse sickness virus (AHSV1 through AHSV9): agar gel immunodiffusion (AGID), indirect fluorescent antibody (IFA), complement fixation (CF), virus neutralization (VN), and enzyme-linked immunosorbent assay (ELISA). The 5 tests were also compared using a panel of field samples, convalescent equine sera with antibodies to domestic equine viral diseases, and sera from horses awaiting export. The ELISA described in this paper was group specific. It did not require calibration with a standard positive serum but did yield elevated values with negative sera that were repeatedly frozen and thawed or heat inactivated. The IFA test was sensitive but could not be used on some field sera as the control cells exhibited fluorescence, possibly due to the animal being recently vaccinated with cell culture material. Sixty-two experimental sera were compared by VN, CF, AGID, and ELISA. Forty sera, 10 positive and 30 negative, were correctly classified by the 5 serologic assays. The 22 remaining sera gave mixed reactions. The AGID had no false positive results but had false negative results for up to 20% of the samples, depending upon the comparison. The VN, CF, and ELISA were similar in their variability. The length of time that virus could be recovered from a viremic blood sample was compared in an evaluation of storage methods for virus isolation samples. Washed erythrocytes were held at 4 C, washed erythrocytes plus stabilizer were held at −70 C, and blood that was drawn into a preservative (oxalate/phenol/glycerol) was held at 4 C. Virus was isolated for 12 months from a sample stored as washed erythrocytes at 4 C but for only 6 months from the same blood stored by the other 2 methods.

The Ethylene Oxide Product Test of Sterility: Limitations and Interpretation of Results

2021 ◽  
Vol 55 (s3) ◽  
pp. 45-56
Author(s):  
Michael Sadowski ◽  
Clark Houghtling ◽  
Sopheak Srun ◽  
Tim Carlson ◽  
Jason Hedrick ◽  
...  
Keyword(s):  
Ethylene Oxide ◽  
False Negative ◽  
Biological Indicator ◽  
Detection Sensitivity ◽  
Study Objective ◽  
Sterility Testing ◽  
Sterility Test ◽  
Negative Results ◽  
Frequency Detection ◽  
Product Test

Abstract The ethylene oxide (EO) product test of sterility (ToS) can be conducted to comply with ANSI/AAMI/ISO 11135:2014 for the generation of data to demonstrate the appropriateness of the biological indicator (BI) that is used to develop and qualify the EO sterilization process. Clause D.8.6 of 11135 provides an option to perform a sublethal EO process, followed by conducting a product ToS, performing sterility testing of BIs from the process challenge device, and comparing the test results. Certain limitations for the EO product ToS should be considered when conducting studies that feature the use of this test, in order to support compliance with this requirement. Limitations for any sterility test include sample size, testing frequency, detection sensitivity, and/or the potential for false-positive/false-negative results, each of which must be recognized and well understood in order to support compliance with the standard. In addition, the experimental design of any study featuring the use of a sterility test should be carefully developed to ensure the generation of scientifically sound results and conclusions to support the study objective.

scholarly journals Polymerase Chain Reaction-Based Detection of Spiroplasma citri Associated with Citrus Stubborn Disease

Plant Disease ◽  
2008 ◽  
Vol 92 (2) ◽  
pp. 253-260 ◽  
Author(s):  
Raymond K. Yokomi ◽  
Alexandre F. S. Mello ◽  
Maria Saponari ◽  
Jacqueline Fletcher
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Disease Incidence ◽  
Chain Reaction ◽  
Spiroplasma Citri ◽  
Field Samples ◽  
Polymerase Chain ◽  
Individual Trees ◽  
Citrus Stubborn Disease

Polymerase chain reaction (PCR)-based detection of citrus stubborn disease was improved using primers based on sequences of the P89 putative adhesin gene and the P58 putative adhesin multigene of Spiroplasma citri. Real-time PCR also was developed with detection limits estimated to be between 10–4 and 10–4 ng by serial dilution of a recombinant S. citri plasmid into DNA extracts from healthy Madam Vinous sweet orange. PCR for the detection of S. citri by these new primers was validated by comparing culturing of the pathogen, the traditional method of diagnosis, with PCR assays from samples taken from two citrus plots in Kern County, CA. Fruit columella was collected from 384 and 377 individual trees in each of two fields, respectively; one portion was used for culturing and the other for DNA extraction and PCR. PCR results matched those of culturing 85 to 100% of the time depending on the primers used. More importantly, PCR detected S. citri from culture-negative trees in 5 to 15% of the cases, suggesting that PCR performed as well or better than culturing for detection of S. citri in field samples. Real-time PCR proved to be the best method for detection. Differential reaction of the samples to the P58 primer pairs suggested that two populations of S. citri occur in historical and present-day field isolates. Citrus stubborn disease incidence was estimated to be 58.3 and 3.7% in the two orchards. The results presented here support the use of PCR for reliable detection of S. citri in field trees.

scholarly journals LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0258263
Author(s):  
Elizabeth C. Stahl ◽  
Allan R. Gopez ◽  
Connor A. Tsuchida ◽  
Vinson B. Fan ◽  
Erica A. Moehle ◽  
...  
Keyword(s):  
Large Scale ◽  
Local Community ◽  
False Negative ◽  
Cost Effective ◽  
Detection Sensitivity ◽  
N Gene ◽  
Negative Results ◽  
Clinical Surveillance ◽  
Sample Pooling ◽  
Wastewater Samples

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a 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 (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

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