scholarly journals SARS-CoV-2 Protein in Wastewater Mirrors COVID-19 Prevalence.

2020 ◽  
Author(s):  
Nafisa Neault ◽  
Aiman T. Baig ◽  
Tyson E. Graber ◽  
Patrick M. D'Aoust ◽  
Elisabeth Mercier ◽  
...  
Keyword(s):  
Rna Virus ◽  
Viral Rna ◽  
Protein Biomarkers ◽  
Chain Reaction ◽  
Epidemiological Tool ◽  
Rna Fragments ◽  
Pcr Method ◽  
Fecal Content ◽  
Polymerase Chain ◽  
Low Levels

The COVID-19 pandemic has given rise to diverse approaches to track infections. The causative agent, SARS-CoV-2 is a fecally-shed RNA virus, and many groups have assayed wastewater for viral RNA fragments by quantitative reverse transcription polymerase chain reaction (qRT-PCR) as a proxy of COVID-19 prevalence in the community. Most groups report low levels of viral RNA that often skirt the methods theoretical limits of detection and quantitation. Here, we demonstrate the presence of SARS-CoV-2 structural proteins in wastewater using traditional immunoblotting and quantitate them from wastewater solids using an immuno-linked PCR method called Multiplex Paired-antibody Amplified Detection (MPAD). In this longitudinal study, we corrected for stochastic variability inherent to wastewater-based epidemiology using multiple fecal content protein biomarkers. These normalized SARS-CoV-2 protein data correlated well with public health metrics. Our method of assaying SARS-CoV-2 protein from wastewater represents a promising and sensitive epidemiological tool to assess prevalence of fecally-shed pathogens in the community.

scholarly journals Polymerase chain reaction (PCR) for the rapid detection of Salmonella in desiccated coconut (DC)

CORD ◽  
2007 ◽  
Vol 23 (2) ◽  
pp. 7
Author(s):  
Jayaratne, D.L.
Keyword(s):  
Polymerase Chain Reaction ◽  
Genomic Dna ◽  
Chain Reaction ◽  
Specific Primers ◽  
Routine Testing ◽  
Cultural Methods ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Low Levels ◽  
M1 Type

This study was carried out to develop a polymerase chain reaction (PCR) protocol for the detection of Salmonella in desiccated coconut (DC). For the PCR detection of Salmonella, genomic DNA was extracted using the ‘boiling lyses’ method and the reaction was carried out with Salmonella genus specific primers enabled to amplify 457bp sequence covering invA and invE genes. Samples of DC produced in mills already tested for Salmonella using conventional cultural methods gave identical results with the present PCR method indicating its suitability for adoption in routine testing.  The sensitivity checked using DNA extracted from artificially inoculated DC with serially diluted inoculum of Salmonella M1 type showed that the developed PCR method can be used to detect very low levels of contamination of Salmonella as low as 4 CFU/g in DC.  The method described here reduces the testing and detection time from 6 days to 24 hours ensuring exporters to obtain Salmonella test reports just prior to shipment. 

Identification of Bacteroides forsythus in Subgingival Dental Plaque with the Aid of a Rapid PCR Method

1997 ◽  
Vol 76 (7) ◽  
pp. 1376-1380 ◽  
Author(s):  
J.H. Meurman ◽  
J. Wahlfors ◽  
A. Korhonen ◽  
P. Alakuijala ◽  
P. Väisänen ◽  
...  
Keyword(s):  
Dental Plaque ◽  
Subgingival Plaque ◽  
Chain Reaction ◽  
Rapid Pcr ◽  
Microbiological Methods ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Pre Treatment ◽  
Culture Positive ◽  
Positive Results

Bacteroides forsythus has been shown to be prevalent among patients with periodontitis. Conventional microbiological methods used to identify this bacterium, however, are laborious and time-consuming and are therefore not well-suited for screening purposes. We have developed a polymerase chain-reaction (PCR) method which is rapid, specific, and simple to perform and does not require other sample pre-treatment except a brief centrifugation. This method was applied to the detection of B. forsythus in subgingival plaque of 58 periodontitis patients. When compared with the results of conventional culturing, the PCR method always confirmed the culture-positive results, while none of the PCR negative samples was shown to be culture-positive. The PCR method appeared to give more than double the number of samples positive for B. forsythus than culturing (89.7% vs. 37.9%). The analysis requires less than 4 hrs to perform, and is specific only to B. forsythus and sensitive enough to detect fewer than 5 bacteria.

scholarly journals Eliminating false positives in a qPCR assay for the detection of the uidA gene in Escherichia coli

2013 ◽  
Vol 11 (3) ◽  
pp. 382-386 ◽  
Author(s):  
Richard Kibbee ◽  
Natalie Linklater ◽  
Banu Örmeci
Keyword(s):  
Escherichia Coli ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Uida Gene ◽  
Chain Reaction ◽  
Taq Polymerase ◽  
E Coli ◽  
Gene Copies ◽  
Polymerase Chain ◽  
Low Levels

Due to contaminant Escherichia coli DNA present in recombinant Taq polymerase reagents, it is not possible to reliably detect low levels of E. coli in samples using the quantitative polymerase chain reaction (qPCR) assay. Native Taq polymerase was successfully used in this study to detect five uidA gene copies (5 fg of genomic DNA) of the uidA gene.

scholarly journals Diversity ofLeptospiraspp. in Rats and Environment from Urban Areas of Sarawak, Malaysia

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Chai Fung Pui ◽  
Lesley Maurice Bilung ◽  
Kasing Apun ◽  
Lela Su’ut
Keyword(s):  
Polymerase Chain Reaction ◽  
Water Samples ◽  
Urban Areas ◽  
Environmental Samples ◽  
Soil Samples ◽  
Chain Reaction ◽  
Prevalence Studies ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Soil And Water

Various prevalence studies onLeptospirain animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophyticLeptospirain selected animals and environments. This study was therefore conducted to detectLeptospiraspp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. PathogenicLeptospirawas present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. IntermediateLeptospirawas present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. SaprophyticLeptospirawas present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76Leptospiraspp. were isolated. Based on DNA sequencing, the dominantLeptospiraspp. circulating in urban areas of Sarawak are pathogenicLeptospira noguchii, intermediateLeptospira wolffiiserovar Khorat, and saprophyticLeptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence ofLeptospiraspp. from rats and the environment, with dominant local serovars in urban areas of Sarawak.

scholarly journals Bovine tuberculosis: Diagnosis by PCR technique in bovine milk samples

2013 ◽  
Vol 37 (2) ◽  
pp. 156-160
Author(s):  
Mohammed J. Alwan
Keyword(s):  
Bovine Milk ◽  
Culture Method ◽  
Bacterial Isolation ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Culture Methods ◽  
Milk Samples ◽  
Tuberculosis Diagnosis ◽  
Pcr Method ◽  
Polymerase Chain

     The aim of this study was to determine the percentage of borne tuberculin infection in milk sample by using PCR.  (102) milk samples were collected from  cows , AL-Dejella (39) samples,  AL-Suara (20) samples cow station, AL-Fthalia (20) samples,  AL-Azezia (11) samples and AL-Twarege (12) samples (Iraq) during the period  July 10th   2010 to  Nov.30th   2010. The samples were examined by direct smear stained by Ziehle-Neelson stain, culture methods and confirmed the isolates by Polymerase Chain Reaction (PCR) assay. The results showed that  5, 102 (4.9%) milk samples were M. bovis positive that were detected by direct milk smear method and 10 out 102(9.8%) M.bovis +ve were detected by culture method and PCR assay. The results also showed that high percentage of bacterial isolates from milk samples AL-Dejella city show (12.8%) by culturing and PCR method followed by AL-Suara (10%), AL-Fthelia (10%), Al-Twarege (8.3%) but no bacterial isolation was recorded in AL-Azezia milk samples. This study concluded that M.bovis infection was spreading in dairy cow within the mentioned areas and PCR was more sensitive, rapid, and accurate technique for M.bovis infection diagnosis.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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