scholarly journals The Molecular Diagnosis Protocols of New Coronavirus (COVID-19); Specificity and Sensitivity an Overview

2020 ◽  
pp. 14-22
Author(s):  
Abdullah Ahmed Hama ◽  
Othman Abdulrahman Mohammed ◽  
Fatima Mahmud Ali ◽  
Osama Hamid Shareef ◽  
Sardar Muhammad Wli ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Acute Respiratory Tract Infection ◽  
Health Concern ◽  
Specific Primers ◽  
Viral Pathogens ◽  
Control Plan ◽  
Infected People ◽  
Specificity And Sensitivity ◽  
Polymerase Chain ◽  
Etiological Agents

Acute respiratory tract infection is a common public health concern worldwide a new emerging contagious virus (COVID-2019) or SARSC- 2 causing a pandemic pneumonia outbreak, The main transmission route of this virus is through droplets from respiratory made during sneezing or coughing of infected people like the recent viral infection of severe acute respiratory syndrome (SARS-CoV1) and the Middle East respiratory syndrome (MERS). Many epidemiological factors have a crucial role in promoting the transmission of the COVID-2019 that makes the disease as an emerging and global alarming against this new coronavirus. Early diagnosis of the etiological agents is critical for appropriate management, controlling plan, protection, and treatment. The new outbreak of COVID-19 can be detected by different molecular protocols. Quantitative polymerase chain reaction (qPCR) is the recommended technique used with varied sensitivity due to primers variation and specimen type. The reliable, high specific and sensitive diagnosis protocols are necessary for an emerging control plan. This study will review and explore the most available methods of molecular identification and primers for the diagnosis of the new coronavirus (COVID-19). This review will also open the new clues to develop and select appropriate diagnosis panel and specific primers for new coronavirus. In conclusion of this review, the RNA dependent RNA polymerase (RdRp) and RdRp/Hel protocols will be valuable to distinguish the  COVID-19 from the SARS-CoV and the other respiratory viral pathogens.

scholarly journals Comparison of endocervical swabs to cultured isolates for the detection of antimicrobial resistance determinants in Neisseria gonorrhoeae

2021 ◽  
pp. 40-46
Author(s):  
G Oree ◽  
M Naicker ◽  
HC Maise ◽  
NS Abbai
Keyword(s):  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
South African ◽  
Quantitative Polymerase Chain Reaction ◽  
Research Direction ◽  
Health Concern ◽  
Penicillin G ◽  
Future Research ◽  
Resistance To Penicillin ◽  
Polymerase Chain

Background: The global emergence of antimicrobial resistance (AMR) in Neisseria gonorrhoeae to various antibiotics is a public health concern. To date, there have been no published South African studies that have compared the primary swab to the cultured isolates for the detection of N. gonorrhoeae AMR determinants. This study provides data on such a comparison. Methods: Paired endocervical swabs were collected from 307 pregnant women. The first swab was stored in an Amies charcoal transport media for culture assessment and the second swab was used for the molecular detection of resistant determinants. Specific targets (genes/plasmids/mutations) associated with resistance to penicillin, tetracycline, ciprofloxacin, spectinomycin, cefixime, azithromycin and ceftriaxone were detected from both the cultured isolates and the endocervical swabs. Results: Of the 307 samples tested in this study, only six samples tested positive for culture. A total of 24 samples tested positive for N. gonorrhoeae with the quantitative polymerase chain reaction (qPCR) assay. The six samples which tested positive for culture fell within the qPCR positives group. Since this study was designed to directly compare the culture swabs to the endocervical swabs for the detection of AMR determinants, the current analysis included only the six culture samples and six paired endocervical swab samples (n = 6). All six isolates were resistant to tetracycline and penicillin G while five of the six isolates were resistant to ciprofloxacin. All isolates were susceptible to the remaining antimicrobials. There was a 100% correlation between the cultured isolates and endocervical swabs for detecting the specific AMR determinants, conferring resistance to tetracycline, penicillin G and ciprofloxacin. Conclusion: Based on the findings of this study, tracking emerging patterns of resistance from the molecular level using only the endocervical swabs may serve as an attractive future research direction.

scholarly journals Two-Stage Adaptive Pooling with RT-qPCR for COVID-19 Screening

2020 ◽  
Author(s):  
Anoosheh Heidarzadeh ◽  
Krishna Narayanan
Keyword(s):  
Polymerase Chain Reaction ◽  
State Of The Art ◽  
Quantitative Polymerase Chain Reaction ◽  
Group Testing ◽  
Soft Information ◽  
Chain Reaction ◽  
Two Stage ◽  
Infected People ◽  
Adaptive Schemes ◽  
Polymerase Chain

AbstractWe propose two-stage adaptive pooling schemes, 2-STAP and 2-STAMP, for detecting COVID-19 using real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) test kits. Similar to the Tapestry scheme of Ghosh et al., the proposed schemes leverage soft information from the RT-qPCR process about the total viral load in the pool. This is in contrast to conventional group testing schemes where the measurements are Boolean. The proposed schemes provide higher testing throughput than the popularly used Dorfman’s scheme. They also provide higher testing throughput, sensitivity and specificity than the state-of-the-art non-adaptive Tapestry scheme. The number of pipetting operations is lower than state-of-the-art non-adaptive pooling schemes, and is higher than that for the Dorfman’s scheme. The proposed schemes can work with substantially smaller group sizes than non-adaptive schemes and are simple to describe. Monte-Carlo simulations using the statistical model in the work of Ghosh et al. (Tapestry) show that 10 infected people in a population of size 961 can be identified with 70.86 tests on the average with a sensitivity of 99.50% and specificity of 99.62%. This is 13.5x, 4.24x, and 1.3x the testing throughput of individual testing, Dorfman’s testing, and the Tapestry scheme, respectively.

Molecular typing ofGiardia duodenalisin humans in Queensland – first report of Assemblage E

Parasitology ◽  
2017 ◽  
Vol 144 (9) ◽  
pp. 1154-1161 ◽  
Author(s):  
ALIREZA ZAHEDI ◽  
DANIEL FIELD ◽  
UNA RYAN
Keyword(s):  
Molecular Typing ◽  
Quantitative Polymerase Chain Reaction ◽  
Giardia Duodenalis ◽  
Protozoan Parasite ◽  
Mixed Infections ◽  
Chain Reaction ◽  
Specific Primers ◽  
First Report ◽  
Polymerase Chain ◽  
Assemblage B

SUMMARYLittle is known about the genetic diversity of the protozoan parasite,Giardia duodenalis,infecting humans in Queensland, Australia. The present study typed 88 microscopicallyGiardia-positive isolates using assemblage-specific primers at the triose phosphate isomerase (tpi) gene and sequenced a subset of isolates at the glutamate dehydrogenase (gdh) gene (n= 30) andtpilocus (n= 27). Using thetpi-assemblage specific primers,G. duodenalisassemblage A and assemblage B were detected in 50% (44/88) and 38·6% (34/88) of samples, respectively. Mixed infections with assemblages A and B were identified in 4·5% (4/88) and assemblage E was identified in 6·8% (6/88) of samples. Sequence analysis at thegdhandtpiloci also confirmed the presence of assemblage E in these isolates. Cyst numbers per gram of feces (g−1) were determined using quantitative polymerase chain reaction and of the isolates that were typed as assemblage E, cyst numbers ranged 13·8–68·3 × 106cysts g−1. This is the first report of assemblage E in humans in Australia, indicating that in certain settings, this assemblage may be zoonotic.

scholarly journals Evaluation of quantitative polymerase chain reaction assays targeting Mycobacterium avium, M. intracellulare, and M. avium subspecies paratuberculosis in drinking water biofilms

2014 ◽  
Vol 13 (1) ◽  
pp. 131-139 ◽  
Author(s):  
Eunice C. Chern ◽  
Dawn King ◽  
Richard Haugland ◽  
Stacy Pfaller
Keyword(s):  
Polymerase Chain Reaction ◽  
Drinking Water ◽  
Mycobacterium Avium ◽  
Dna Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Specificity And Sensitivity ◽  
Polymerase Chain ◽  
Mycobacterium Avium Subsp Paratuberculosis

Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples.

scholarly journals Efficacy of a Modified Polymerase Chain Reaction Assay for Detection of Ehrlichia Canis infection

2000 ◽  
Vol 12 (5) ◽  
pp. 456-459 ◽  
Author(s):  
John S. Mathew ◽  
Sidney A. Ewing ◽  
Jerry R. Malayer ◽  
Joseph C. Fox ◽  
Katherine M. Kocan
Keyword(s):  
Polymerase Chain Reaction ◽  
Ehrlichia Canis ◽  
New Method ◽  
Conventional Pcr ◽  
Serologic Test ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Primers ◽  
Specificity And Sensitivity ◽  
Polymerase Chain

Detection of Ehrlichia canis in acutely infected and convalescent dogs is important for effective treatment and control. However, accurate detection has been difficult to achieve, in part because dogs that have been treated therapeutically often remain seropositive for extended periods. A new method, polymerase chain reaction (PCR) assay using biotinylated E. canis-specific primers (PCR-BP), was developed for detection of E. canis. Four dogs experimentally infected with E. canis by intravenous inoculation of whole blood from carrier dogs and 2 naturally infected convalescent carriers were used to compare the specificity and sensitivity of the new method with that of microscopy/blood smear evaluation, serologic test, and conventional PCR assay using E. canis-specific primers. In experimentally infected animals, infection was detected as early as 7 days postexposure using PCR-BP. Although the 2 naturally infected dogs were positive by serologic test and PCR-BP, both were negative by conventional PCR. Results suggest that the new method is a sensitive assay for detection of E. canis infection. In addition, results were obtained more rapidly than with other PCR-based assays.

Somatic coliphages as surrogates for enteroviruses in sludge hygienization treatments

2016 ◽  
Vol 73 (9) ◽  
pp. 2182-2188 ◽  
Author(s):  
Julia Martín-Díaz ◽  
Raquel Casas-Mangas ◽  
Cristina García-Aljaro ◽  
Anicet R. Blanch ◽  
Francisco Lucena
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Detection Accuracy ◽  
Chain Reaction ◽  
Viral Pathogens ◽  
Somatic Coliphages ◽  
Treatment Processes ◽  
Novel Treatment ◽  
Polymerase Chain ◽  
Viral Indicators ◽  
Relationship Of

Conventional bacterial indicators present serious drawbacks giving information about viral pathogens persistence during sludge hygienization treatments. This calls for the search of alternative viral indicators. Somatic coliphages’ (SOMCPH) ability for acting as surrogates for enteroviruses was assessed in 47 sludge samples subjected to novel treatment processes. SOMCPH, infectious enteroviruses and genome copies of enteroviruses were monitored. Only one of these groups, the bacteriophages, was present in the sludge at concentrations that allowed the evaluation of treatment's performance. An indicator/pathogen relationship of 4 log10 (PFU/g dw) was found between SOMCPH and infective enteroviruses and their detection accuracy was assessed. The obtained results and the existence of rapid and standardized methods encourage the inclusion of SOMCPH quantification in future sludge directives. In addition, an existing real-time quantitative polymerase chain reaction (RT-qPCR) for enteroviruses was adapted and applied.

scholarly journals ProbeSpec: batch specificity testing and visualization of oligonucleotide probe sets implemented in ARB

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 1901
Author(s):  
Tim Kahlke ◽  
Paavo Jumppanen ◽  
Ralf Westram ◽  
Guy C.G. Abell ◽  
Levente Bodrossy
Keyword(s):  
Sensitivity And Specificity ◽  
Quantitative Polymerase Chain Reaction ◽  
High Specificity ◽  
Cost Effective ◽  
Marker Genes ◽  
Oligonucleotide Probes ◽  
Cross Hybridization ◽  
Specificity And Sensitivity ◽  
Novel Taxa ◽  
Polymerase Chain

High-throughput molecular methods such as quantitative polymerase chain reaction (qPCR) and environmental microarrays are cost-effective methods for semi-quantitative assessment of bacterial community structure and the identification of specific target organisms. Both techniques rely on short nucleotide sequences, so-called oligonucleotide probes, which require high specificity to the organisms in question to avoid cross-hybridization with non-target taxa. However, designing oligonucleotide probes for novel taxa or marker genes that show sufficient phylogenetic sensitivity and specificity is often time- and labor-intensive, as each probe has to be in-silico tested for its specificity and sensitivity. Here we present ProbeSpec, to our knowledge the first batch sensitivity and specificity estimation and visualization tool for oligonucleotide probes integrated into the widely used ARB software. Using ProbeSpec’s interactive “mismatch threshold” and “clade marked threshold” we were able to reduce the development time of highly specific probes for a recently published environmental oligonucleotide microarray from several months to one week.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1759-1767 ◽  
Author(s):  
Kyu-Tae Kim ◽  
Kristin Baird ◽  
Joon-Young Ahn ◽  
Paul Meltzer ◽  
Michael Lilly ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Quantitative Polymerase Chain Reaction ◽  
Colony Growth ◽  
Dominant Negative ◽  
Internal Tandem Duplication ◽  
Downstream Target ◽  
Before And After ◽  
Flt3 Expression ◽  
Polymerase Chain ◽  
Expression Microarrays

AbstractConstitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukemogenesis, and their presence is associated with poor prognosis in acute myeloid leukemia (AML). To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild-type FLT3 expression. Microarrays were used with RNA harvested before and after inhibition of FLT3 signaling. Pim-1 was found to be one of the most significantly down-regulated genes upon FLT3 inhibition. Pim-1 is a proto-oncogene and is known to be up-regulated by signal transducer and activator of transcription 5 (STAT5), which itself is a downstream target of FLT3 signaling. Quantitative polymerase chain reaction (QPCR) confirmed the microarray results and demonstrated approximately 10-fold decreases in Pim-1 expression in response to FLT3 inhibition. Pim-1 protein also decreased rapidly in parallel with decreasing autophosphorylation activity of FLT3. Enforced expression of either the 44-kDa or 33-kDa Pim-1 isotypes resulted in increased resistance to FLT3 inhibition-mediated cytotoxicity and apoptosis. In contrast, expression of a dominant-negative Pim-1 construct accelerated cytotoxicity in response to FLT3 inhibition and inhibited colony growth of FLT3/ITD-transformed BaF3 cells. These findings demonstrate that constitutively activated FLT3 signaling up-regulates Pim-1 expression in leukemia cells. This up-regulation contributes to the proliferative and antiapoptotic pathways induced by FLT3 signaling. (Blood. 2005;105: 1759-1767)

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