scholarly journals Comparison of in-house SARS-CoV-2 genome extraction procedures. A need for COVID-19 pandemic

2021 ◽  
Author(s):  
Gabriel Martin ◽  
Susana Rojo-Alba ◽  
Cristian Castelló-Abietar ◽  
Fátima Abreu-Salinas ◽  
Isabel Costales ◽  
...  
Keyword(s):  
Reference Method ◽  
Detection Methods ◽  
Automatic Method ◽  
Rt Pcr ◽  
Extraction Procedures

Abstract Purpose: Among the methods used to diagnose COVID-19, those based on genomic detection by q(RT)-PCR are the most sensitive. To perform these assays, a previous genome extraction of the sample is required. The dramatic increase in the number of SARS-CoV-2 detection assays has increased the demand for extraction reagents hindering the supply of commercial reagents. Homemade reagents and procedures could be an alternative. Methods: Nasopharyngeal samples were extracted by seven different methods as well as the automatic method MagNaPure96, to detect SARS-CoV-2. Results: All protocols show sensitivity higher than 87%, in comparison with reference method, for detecting SARS-CoV-2 as well as human β- globin. Conclusions: Our results support that these procedures, using common and cheap reagents, are effective to extract RNA (from SARS-CoV-2) or DNA (from human β-globin) genome from nasopharyngeal swabs. Furthermore, these procedures could be easily adopted by routine diagnostic laboratories to implement detection methods to help to fight against COVID-19 pandemic.

Comparative Evaluation of a Phage Protein Ligand Assay with Real-Time PCR and a Reference Method for the Detection of Escherichia coli O157:H7 in Raw Ground Beef and Trimmings

2011 ◽  
Vol 74 (1) ◽  
pp. 6-12 ◽  
Author(s):  
F. SAVOYE ◽  
P. FENG ◽  
C. ROZAND ◽  
M. BOUVIER ◽  
A. GLEIZAL ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Ground Beef ◽  
Reference Method ◽  
Enterohemorrhagic Escherichia Coli ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Rt Pcr ◽  
Raw Meat ◽  
E Coli

Enterohemorrhagic Escherichia coli O157:H7 is an important pathogen associated with infections caused by consumption of undercooked raw meat. Sensitive and rapid detection methods for E. coli O157:H7 are essential for the meat industry to ensure a safe meat supply. This study was conducted to compare the sensitivity of the VIDAS ultraperformance E. coli test (ECPT UP) with a noncommercial real-time (RT) PCR method and the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) reference method for detecting E. coli O157:H7 in raw ground beef. Optimal enrichment times and the efficacy of testing different types of raw meat, either as individual samples (25 g) or as composites (375 g), were examined. For 25-g samples of each type of raw ground beef tested, 6 h of enrichment was sufficient for both the VIDAS ECPT UP and RT-PCR methods, but for 375-g samples, 24 h of enrichment was required. Both the VIDAS ECPT UP and RT-PCR methods produced results similar to those obtained with the USDA-FSIS reference method after 18 to 24 h of enrichment. The primer specificity of the RT-PCR assay and the highly specific phage ligand used in the VIDAS ECPT UP for target recognition enabled the detection of low levels of E. coli O157:H7 in 25 g of various types of raw ground beef. The tests also allowed the detection of E. coli O157:H7 in composite raw ground beef and trimmings in samples of up to 375 g.

scholarly journals A multi-laboratory comparison of two molecular methods for the detection of toxigenic Clostridium difficile

2016 ◽  
Vol 10 (01) ◽  
pp. 62-67 ◽  
Author(s):  
Diane C Halstead ◽  
Joan Abid ◽  
Lynne Sloan ◽  
Diana Meza ◽  
Daphne Ramsey-Walker ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Molecular Characterization ◽  
Regulatory Gene ◽  
Reference Method ◽  
Molecular Methods ◽  
Detection Methods ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Toxigenic Culture

Introduction: Diarrheal disease due to toxigenic Clostridium difficile (CD) accounts for an increased number of hospitalizations and deaths each year. Published guidelines recommend reflex testing of CD antigen-positive samples to molecular testing or testing samples directly by a molecular assay. This multicenter study was designed to compare the accuracy of two different molecular methods targeting different CD genes: Xpert C. difficile Epi RUO RT-PCR assay (XPCR) which targets toxin B (Cepheid, Sunnyvale, CA) and a laboratory-developed PCR (LDPCR) which targets mutations in the tcdC regulatory gene. Methodology: Two molecular methods for toxigenic CD detection, the Xpert C. difficile Epi RUO RT-PCR assay (XPCR) [Cepheid, Sunnyvale, CA] and a laboratory-developed PCR assay (LDPCR) were compared to a consensus gold standard (CGS) or toxigenic culture (TC) as the reference method. A subset of specimens was subjected to additional molecular characterization of toxigenic CD. Results: Both molecular methods were >90% sensitive for CD detection. Discordant results were noted when molecular test results were compared to non-molecular methods. Supplemental molecular characterization illustrated inherent difficulties in comparisons using different molecular methods for CD. Conclusion: Laboratories may consider using multiple CD detection methods or combinations of methods, including molecular detection for rapid and accurate diagnosis of CD, as driven by best practices for the respective healthcare environment. Laboratories must be aware of intrinsic differences when comparing performance characteristics of different molecular assays.

scholarly journals Molecular Diagnosis in Differentiating Active and Inactive Forms of Hepatitis B Virus Carriers

2018 ◽  
Vol 26 (10) ◽  
pp. 41-45
Author(s):  
Salah Tofik Jalal Balaky ◽  
Saeed Ghulam Hussain ◽  
Amer Ali Khaleel ◽  
Furat Tahseen Sabeer ◽  
Ahang Hasan Mawlood
Keyword(s):  
Nucleic Acid ◽  
Hepatitis B ◽  
Cross Sectional Study ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Detection Methods ◽  
Rt Pcr ◽  
Cross Sectional ◽  
Blood Samples ◽  
Acid Test

Background & objectives: Introducing a nucleic acid test program is aimed to diagnose and reduces the risk of viral infection or transmission. DNA assay for HBV can detect infection in the windows period, chronic occult infection and can discriminate between active and inactive HBV infection. This cross-sectional study designed to diagnose, analyze HBV infection and to differentiate active from inactive infection based on viral DNA detection. Methods: Blood samples were collected from 256 patients previously diagnosed on the clinical ground as hepatitis B seropositive in Erbil Central Lab. The viral nucleic acid quantitative assessment was done for the collected samples using RT-PCR. Q-square was performed for statistical analysis. Results: Out of 256 collected blood samples 93 (36.3%) showed HBV-DNA positive titers above 50 IU/ml. Among positive subjects, 67 (72.04%) was categorized as inactive carriers (˂ 2000-20.000 IU/ml HBV-DNA titers). Conclusions: The data produced from this study confirmed the importance of the RT-PCR technique in sensitivity and reliability as a superior diagnostics tool specifically in differentiating active from inactive HBV carriers.

scholarly journals RT-PCR for COVID-19: Diagnosis Made Easy

BioMedica ◽  
2020 ◽  
Vol 36 (2S) ◽  
pp. 115-120
Author(s):  
Osheen Sajjad ◽  
Aiman Shahzad ◽  
Saqib Mahmood
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Detection Method ◽  
Current Knowledge ◽  
Detection Methods ◽  
Rt Pcr ◽  
Corona Virus ◽  
Positive Sense ◽  
Affected Person ◽  
Sampling Procedures

<p>Coronavirus disease COVID-19, caused by Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV2), is highly contagious and has been a pandemic since March 2020. The SARS-CoV-2 is an enveloped, single-stranded, positive-sense RNA viruswhich spreadsthrough air droplets by sneezing and coughing from affected person. The diagnosis of the COVID-19 remains a challenge to the scientists since the genome of the SARS-CoV-2 was novel and varying. Various studies have reported the validated procedures for sampling and the detection method of SARS-CoV-2. This mini-review provides a brief introduction of the SARS-CoV-2 features and the current knowledge for the recommended COVID19 detection methods including sampling procedures and real time SARS-CoV-2 genome detection.</p>

scholarly journals Characterization and Use of an Antibody Detecting the CBFβ-SMMHC Fusion Protein in inv(16)/t(16;16)-Associated Acute Myeloid Leukemias

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1882-1890
Author(s):  
David S. Viswanatha ◽  
I.-Ming Chen ◽  
Pu Paul Liu ◽  
Marilyn L. Slovak ◽  
Cathy Rankin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Type A ◽  
Detection Methods ◽  
Rt Pcr ◽  
Cytogenetic Abnormalities ◽  
Permeabilized Cells ◽  
Flow Cytometric ◽  
Acute Myeloid

The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFβ-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFβ-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFβ-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR–negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFβ-SMMHC fusion proteins. Molecular characterization of one RT-PCR–positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, −0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR–positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.

scholarly journals Virological and Parasitological Characterization of Mini-LEWE Minipigs Using Improved Screening Methods and an Overview of Data on Various Minipig Breeds

2021 ◽  
Vol 9 (12) ◽  
pp. 2617
Author(s):  
Sabrina Halecker ◽  
Julia Metzger ◽  
Christina Strube ◽  
Ludwig Krabben ◽  
Benedikt Kaufer ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Massachusetts General Hospital ◽  
Hepatitis E ◽  
Human Cells ◽  
Gastrointestinal Parasites ◽  
Endogenous Retroviruses ◽  
Detection Methods ◽  
Screening Methods ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear

Minipigs play an important role in biomedical research and have also been used as donor animals in xenotransplantation. To serve as a donor in xenotransplantation, the animals must be free of potential zoonotic viruses, bacteria and parasites. Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated as most of the other pig viruses can. PERV-A and PERV-B infect human cells in cell culture and are integrated in all pigs, whereas PERV-C infects only pig cells and it is found in many, but not all pigs. Minipigs are known for a high prevalence of recombinant PERV-A/C viruses able to infect human cells (Denner and Schuurman, Viruses, 2021;13:1869). Here, Mini-LEWE minipigs are screened for the first time for pig viruses including PERV. Peripheral blood mononuclear cells (PBMCs) from 10 animals were screened using PCR-based methods (PCR, RT-PCR, and real-time PCR). In comparison with our previous screening assays, numerous improvements were introduced, e.g., the usage of gene blocks as a PCR standard and foreign RNA to control reverse transcription in RT-PCR. Using these improved detection methods, Mini-LEWE pigs were found to be negative for porcine cytomegalovirus (PCMV), porcine lymphotropic herpesviruses (PLHV-1, -2 and -3), porcine circoviruses (PCV1, 2, 3 and 4), porcine parvovirus (PPV) and hepatitis E virus (HEV). All animals carried PERV-A, PERV-B and PERV-C in their genome. PERV-A/C was not found. In contrast to all other minipig breeds (Göttingen minipigs, Aachen minipigs, Yucatan micropig, Massachusetts General Hospital miniature pigs), Mini-LEWE minipigs have less viruses and no PERV-A/C. Parasitological screening showed that none of the Mini-LEWE minipigs harbored ecto- and gastrointestinal parasites, but at least one animal tested positive for anti-Toxoplasma gondii antibodies.

Universal primers for rapid detection of six pospiviroids in Solanaceae plants using one-step RT-PCR and RT-LAMP

Plant Disease ◽  
2021 ◽  
Author(s):  
Yi-Wen Tseng ◽  
Chien-Fu Wu ◽  
Chia-Hwa Lee ◽  
Chung Jan Chang ◽  
Yuh-Kun Chen ◽  
...  
Keyword(s):  
Potato Spindle Tuber Viroid ◽  
Detection Methods ◽  
Universal Primers ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Highly Sensitive ◽  
One Step ◽  
Tomato Chlorotic Dwarf Viroid ◽  
Minimal Concentration ◽  
Solanaceous Plants

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are currently listed as quarantine pathogens in many countries. Among them, columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd), and tomato planta macho viroid (TPMVd) are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from one femtogram to 10 nanograms, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods currently in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate to screen a large number of solanaceous plants and seeds intended for import and export.

Bacteriophage-Based Enrichment Coupled to Immunochromatographic Strip–Based Detection for the Determination of Salmonella in Meat and Poultry

2007 ◽  
Vol 70 (10) ◽  
pp. 2235-2242 ◽  
Author(s):  
MARK T. MULDOON ◽  
GEORGE TEANEY ◽  
JINGKUN LI ◽  
DALE V. ONISK ◽  
JAMES W. STAVE
Keyword(s):  
Reference Method ◽  
Test Strip ◽  
False Positives ◽  
Food Sources ◽  
Detection Methods ◽  
Immunochromatographic Strip ◽  
Conventional Culture ◽  
Sample Enrichment ◽  
Enrichment Step

Immunochemical-based methods for the detection of Salmonella in food can be complicated by the presence of closely related, immunocrossreactive non-Salmonella species in the sample that may cause false-positive results. To circumvent this problem, specific bacteriophages against immunocrossreactive, non-Salmonella bacteria were used in the sample enrichment step to suppress their growth and improve the performance of an immunochromatographic strip–based detection method for Salmonella. Cross-reactive bacteria were isolated from various food sources and were characterized with a panel of Salmonella somatic O antigen–specific monoclonal antibodies. These cross-reactive bacteria were primarily Citrobacter spp. and Escherichia coli with serology shared with Salmonella serogroups B, D, and F. These bacteria were used as hosts for the isolation of specific lytic bacteriophages. When formulated with the primary enrichment, the bacteriophage cocktail significantly reduced false positives with a broadly reactive immunochromatographic test strip. This was demonstrated in both artificially and naturally contaminated meat. False positives in naturally contaminated beef samples were reduced from 32 of 115 samples tested to zero. In raw meat and poultry with a relatively high bioburden (&gt;105 CFU/g), the use of the bacteriophage-based enrichment procedure gave improved recovery of Salmonella compared with the conventional culture-based reference method. This was observed when coupled to either test strip–based or selective agar–based detection. The use of specific bacteriophages for the control of immunocrossreactive and competitive microflora during the food sample enrichment step provides a new approach for enhancing the performance of both immunological- and cultural-based detection methods.

scholarly journals Detection and Quantification of Influenza Virus Defective Viral Genomes from NGS Datasets Obtained after RT or RT-PCR Product Sequencing

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 84
Author(s):  
Jeremy Boussier ◽  
Sandie Munier ◽  
Bernadette Crescenzo-Chaigne ◽  
Sylvie Behillil ◽  
Vincent Enouf ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Molecular Mechanisms ◽  
Pcr Amplification ◽  
Influenza Viruses ◽  
Detection Methods ◽  
Rt Pcr ◽  
Bioinformatics Pipeline ◽  
Genomic Rnas ◽  
Viral Genomes ◽  
Detection And Quantification

Like most RNA viruses, influenza viruses (IAV) generate defective viral genomes (DVGs) during viral replication. Although there is accumulating evidence of a biological impact of DVGs, the molecular mechanisms leading to their production remain to be unveiled. Various next-generation sequencing (NGS) technologies and detection methods can be used to characterize DVGs. Here, we developed a bioinformatics pipeline called DG-seq to quickly identify and quantify DVGs in influenza viral stocks and compared two processing methods for NGS, with or without PCR amplification. To evaluate the performance of the DG-seq pipeline, we used either synthetic in-vitro-transcribed DVGs mixed with the full set of synthetic full-length genomic RNAs, or biological RNA samples extracted in duplicate from three IAV stocks: mutant viruses with a K635A or a R638A mutation in the PA subunit of the polymerase that impairs viral transcription, and their wild-type (WT) counterpart. Viral genomic RNAs were reverse-transcribed and either directly subjected to Illumina sequencing (RT-seq) or PCR-amplified prior to sequencing (RT-PCR-seq). Both methods displayed a good reproducibility between batches, with a lower detection rate but a more accurate quantification of DVGs in RT-seq samples. The PA mutants produced more DVGs than the WT virus, derived mostly from the polymerase gene segments, but also from the NA and HA segments, suggesting that an imbalance between transcription and replication can promote DVG production. Breakpoints occurred near the segment extremities, with no hotspot identified. Interestingly, we observed short direct A/T-rich repeats adjacent to the breakpoint ends at a significantly higher frequency than in the random case. This work provides the first comparison of DVG detection and quantification from NGS data obtained in the presence or absence of PCR amplification and gives novel insight into the mechanisms of influenza virus DVG production.

scholarly journals Challenges in vitamin D analysis / Izazovi u analizi vitamina D

2012 ◽  
Vol 31 (4) ◽  
pp. 326-332 ◽  
Author(s):  
Mustafa Serteser ◽  
Abdurrahman Coskun ◽  
Tamer C Inal ◽  
Ibrahim Unsal
Keyword(s):  
Vitamin D ◽  
Clinical Laboratory ◽  
Direct Detection ◽  
Reference Method ◽  
Detection Methods ◽  
Binding Assays ◽  
External Quality Assessment Scheme ◽  
Specificity And Sensitivity ◽  
Competitive Protein Binding ◽  
Phosphorus Levels

Summary Vitamin D is an important determinant for the regulation of calcium and phosphorus levels and mineralization of the bone. The most reliable indicator of vitamin D status is the measurement of plasma or serum 25OH-D concentration. Several studies reported discrepancies between the results of assays. These high variabilities in 25OH-D measurements are due to used assay technologies and lack of standardization against the reference materials. Different assays have been employed for the measurement of 25OHD levels: Competitive Protein Binding Assays, immunoassays, direct detection methods. Choosing an assay platform is important both for clinical laboratory professionals and researchers, and several factors affect this process. Recently, liquid chromatography and tandem mass spectrometry is an alternative method to traditional assays and provides higher specificity and sensitivity than many assays; therefore, it has been suggested as a candidate reference method for circulating 25OH-D3. Standardization of methods for the quantification of 25OH-D by using the human-based samples would reduce the inter-method variability. The best way for laboratories to demonstrate the accuracy of their results is by participating in the external quality assessment scheme. Standardization of the assays is also required to provide clinicians with the accurate tools to diagnose hypovitaminosis. In addition, assay-specific decision limits are needed to define appropriate thresholds of treatment.

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