scholarly journals A protocol for the detection of genetic markers in saliva by polymerase chain reaction without a nucleic acid purification step: examples of SARS-CoV-2 and GAPDH markers.

2021 ◽  
Author(s):  
Surya Kannan ◽  
Johan Ericsson ◽  
Nazariy Souchelnytskyi ◽  
Serhiy Souchelnytskyi
Keyword(s):  
Nucleic Acids ◽  
Genetic Markers ◽  
Room Temperature ◽  
Direct Detection ◽  
Sodium Dodecyl ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Purification Step ◽  
Efficient Detection ◽  
Nucleic Acid Purification

Abstract Background: The objective of this study was to develop a protocol for direct use of saliva in tests for genetic markers, without purification of nucleic acids. Currently, diagnostic tests use purified nucleic acids from clinical samples. This purification step adds time, cost, and affects the quality of testing. Multiple attempts to remove the purification step were reported. Results: We report a protocol for the direct detection of genetic markers in saliva. The protocol is based on collection of saliva in a solution containing a detergent and ethanol, and is compatible with isothermal amplification (LAMP), real-time RT-PCR and RT-PCR. SARS-CoV-2 and GAPDH markers were used as reference markers. We observed that mild detergents allow efficient detection of markers (e.g. GAPDH and SARS-CoV-2), while strong detergent, e.g. sodium dodecyl sulfate, inhibited the PCR reaction. Under these conditions, saliva samples can be stored for 24 h at +40C or -180C with preservation of the markers. Storage at room temperature led to deterioration of marker detection. Snap heating of saliva samples at the time of collection, followed by a storage at the room temperature, provided partial protection.Conclusions: The protocol presented in this report describes collection and storage of saliva for direct detection of genetic markers and is compatible with PCR and LAMP tests.

scholarly journals A PROTOCOL FOR THE DETECTION OF GENETIC MARKERS IN SALIVA BY POLYMERASE CHAIN REACTION WITHOUT A NUCLEIC ACID PURIFICATION STEP: EXAMPLES OF SARS-COV-2 AND GAPDH MARKERS

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Surya Kannan ◽  
◽  
Johan Ericsson ◽  
Nazariy Souchelnytskyi ◽  
Serhiy Souchelnytskyi ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acids ◽  
Real Time ◽  
Genetic Markers ◽  
Room Temperature ◽  
Direct Detection ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Introduction. Polymerase chain reaction (PCR)-based diagnostic tests use purifi ed nucleic acids (NAs) from clinical samples. The NAs purifi cation step adds time, cost, and aff ects the quality of testing. The objective of this study was to develop a protocol for direct use of saliva in tests for genetic markers, without purifi cation of nucleic acids. Methods. PCR, real-time RT-PCR and isothermal amplifi cation tests were used for direct detection of genetic markers, without purifi cation of nucleic acids. Results. We report a protocol for the direct detection of genetic markers in saliva. The protocol is based on a collection of saliva in a solution containing a detergent and ethanol and is compatible with isothermal amplifi cation (LAMP), real-time RT-PCR and RT-PCR. SARS-CoV-2 and GAPDH markers were used as reference markers. We observed that mild detergents allow effi cient detection of external reference and intracellular endogenous markers, while strong detergent, e.g. sodium dodecyl sulfate, inhibited the PCR reaction. Under these conditions, saliva samples can be stored for 24 h at +4°C or –18°C with the preservation of markers. Storage at room temperature led to the deterioration of marker detection. Snap heating of saliva samples at the time of collection, followed by storage at room temperature, provided partial protection. Conclusion. The protocol presented in this report describes the collection and storage of saliva for direct detection of genetic markers and is compatible with PCR and LAMP tests.

scholarly journals Sensitive quantitative detection of SARS-CoV-2 in clinical samples using digital warm-start CRISPR assay

2020 ◽  
Author(s):  
Xiong Ding ◽  
Kun Yin ◽  
Ziyue Li ◽  
Maroun M. Sfeir ◽  
Changchun Liu
Keyword(s):  
Room Temperature ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
High Tolerance ◽  
Rt Pcr ◽  
One Pot ◽  
Nucleoprotein Gene ◽  
Warm Start ◽  
Heat Treated

AbstractQuantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is crucial for assessing the infectivity of coronavirus disease 2019 and the efficacy of antiviral drugs. Here, we describe a digital warm-start CRISPR (WS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples. The WS-CRISPR assay combines low-temperature reverse transcription dual-priming mediated isothermal amplification (RT-DAMP) and CRISPR-Cas12a-based detection in one-pot, attributed to the mediation role by pyrophosphatase and phosphorothioated primers. The WS-CRISPR assay is initiated at above 50 °C and overcomes undesired premature target amplification at room temperature, enabling accurate digital nucleic acid quantification. By targeting SARS-CoV-2’s nucleoprotein gene, digital WS-CRISPR assay is able to detect down to 5 copies/μl SARS-CoV-2 RNA in the chip within 90 minutes. It is clinically validated by quantitatively determining 32 clinical swab samples and three clinical saliva samples, showing 100% agreement with RT-PCR results. Moreover, the digital WS-CRISPR assay has been demonstrated to directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction, showing high tolerance to inhibitors. Thus, the digital WS-CRISPR method, as a sensitive and reliable CRISPR assay, facilitates accurate SARS-CoV-2 detection toward digitized quantification.

Real-time PCR Detection of Rhodococcus fascians and Discovery of New Plants Associated with R. fascians in Pennsylvania

2012 ◽  
Vol 13 (1) ◽  
pp. 24 ◽  
Author(s):  
Ekaterina V. Nikolaeva ◽  
Seogchan Kang ◽  
Tracey N. Olson ◽  
SeongHwan Kim
Keyword(s):  
Real Time ◽  
Direct Detection ◽  
Infected Plant ◽  
Clinical Samples ◽  
Specific Method ◽  
Rt Pcr ◽  
Plant Materials ◽  
Rhodococcus Fascians ◽  
Wide Range ◽  
Positive Direct

Rhodococcus fascians is a gram-positive bacterium that causes bacterial fasciation on a wide range of ornamental plants. To address the need for a reliable, sensitive, and specific method for detecting R. fascians in infected plant materials, a real-time (RT) PCR assay was developed. The target for detection was fas-1, a plasmid-borne gene that is essential for virulence. DNAs from all confirmed pathogenic strains of R. fascians consistently tested positive, with detection limit of 30 fg of R. fascians DNA. In repeated PCR experiments with R. fascians pure culture, as few as 2.5 CFU were tested positive. Direct detection of R. fascians from clinical samples of Coreopsis was successful, whereas detection of R. fascians in Chrysanthemum, Pelargonium, Phlox, and Veronica required enrichment on modified D2 (mD2) medium. In the case of geraniums, as few as 102 CFU/100 mg plant tissues were successfully detected after 72 h enrichment on mD2. In total, 115 strains, isolated from 41 different kinds of flowering crops in Pennsylvania greenhouses during 1984-2010, were confirmed to be R. fascians by RT PCR. Geraniums and speedwell were the most frequently submitted clinical samples to Pennsylvania Department of Agriculture. The following are the first report of plants associated with R. fascians in PA: Ajania pacifica, Anemone sp., Aruncus sp., Baptisia sp., Eutrochium maculatum, Helianthemum sp., Lewisia sp., Monarda sp., Osteospermum ecklonis, Rudbeckia nitida, and Saponaria ocymoides. Accepted for publication 30 November 2011. Published 27 February 2012.

scholarly journals Urinary nucleic acids can be concentrated, dried on membranes and stored at room temperature in vacuum bags

2014 ◽  
Author(s):  
Fanshuang Zhang ◽  
Yuan Yuan ◽  
Jianqiang Wu ◽  
Youhe Gao
Keyword(s):  
Nucleic Acids ◽  
Large Scale ◽  
Room Temperature ◽  
Invasive Disease ◽  
The Body ◽  
Clinical Samples ◽  
Good Source ◽  
Disease Biomarkers ◽  
Non Invasive

Urine accumulates traces of changes that occur in the body and can potentially serve as a better biomarker source. Urinary nucleic acids are a promising class of non-invasive disease biomarkers. However, long-term frozen human urine samples are not a good source for the extraction of nucleic acids. In this paper, we demonstrate that urinary nucleic acids can be concentrated, dried on membranes and stored in vacuum bags at room temperature for several months. The amount of total RNA on the membranes after storage at room temperature for three months was unchanged. The levels of miR-16 and miR-21 exhibited no significant differences (P = 0.564, 0.386). This simple and economical method makes the large-scale storage of clinical samples of urinary nucleic acids possible.

scholarly journals Evaluation of the QIAstat-Dx Respiratory SARS-CoV-2 Panel, the First Rapid Multiplex PCR Commercial Assay for SARS-CoV-2 Detection

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Benoit Visseaux ◽  
Quentin Le Hingrat ◽  
Gilles Collin ◽  
Donia Bouzid ◽  
Samuel Lebourgeois ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Limit Of Detection ◽  
Clinical Sample ◽  
Point Of Care ◽  
Sample Pretreatment ◽  
Tracheal Aspirate ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Viral Neutralization ◽  
Efficient Detection

ABSTRACT In the race to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), efficient detection and triage of infected patients must rely on rapid and reliable testing. In this work, we performed the first evaluation of the QIAstat-Dx respiratory SARS-CoV-2 panel (QIAstat-SARS) for SARS-CoV-2 detection. This assay is the first rapid multiplex PCR (mPCR) assay, including SARS-CoV-2 detection, and is fully compatible with a non-PCR-trained laboratory or point-of-care (PoC) testing. This evaluation was performed using 69 primary clinical samples (66 nasopharyngeal swabs [NPS], 1 bronchoalveolar lavage fluid sample [BAL], 1 tracheal aspirate sample, and 1 bronchial aspirate sample) comparing SARS-CoV-2 detection with the currently WHO-recommended reverse transcription-PCR (RT-PCR) (WHO-RT-PCR) workflow. Additionally, a comparative limit of detection (LoD) assessment was performed for QIAstat-SARS and WHO-RT-PCR using a quantified clinical sample. Compatibility of sample pretreatment for viral neutralization or viscous samples with the QIAstat-SARS system were also tested. The QIAstat-Dx respiratory SARS-CoV-2 panel demonstrated a sensitivity comparable to that of the WHO-recommended assay with a limit of detection at 1,000 copies/ml. The overall percent agreement between QIAstat-Dx SARS and WHO-RT-PCR on 69 clinical samples was 97% with a sensitivity of 100% (40/40) and specificity at 93% (27/29). No cross-reaction was encountered for any other respiratory viruses or bacteria included in the panel. The QIAstat-SARS rapid multiplex PCR panel provides a highly sensitive, robust, and accurate assay for rapid detection of SARS-CoV-2. This assay allows rapid decisions even in non-PCR-trained laboratory or point-of-care testing, allowing innovative organization.

scholarly journals Urinary nucleic acids can be concentrated, dried on membranes and stored at room temperature in vacuum bags

2014 ◽  
Author(s):  
Fanshuang Zhang ◽  
Yuan Yuan ◽  
Jianqiang Wu ◽  
Youhe Gao
Keyword(s):  
Nucleic Acids ◽  
Large Scale ◽  
Room Temperature ◽  
Invasive Disease ◽  
The Body ◽  
Clinical Samples ◽  
Good Source ◽  
Disease Biomarkers ◽  
Non Invasive

Urine accumulates traces of changes that occur in the body and can potentially serve as a better biomarker source. Urinary nucleic acids are a promising class of non-invasive disease biomarkers. However, long-term frozen human urine samples are not a good source for the extraction of nucleic acids. In this paper, we demonstrate that urinary nucleic acids can be concentrated, dried on membranes and stored in vacuum bags at room temperature for several months. The amount of total RNA on the membranes after storage at room temperature for three months was unchanged. The levels of miR-16 and miR-21 exhibited no significant differences (P = 0.564, 0.386). This simple and economical method makes the large-scale storage of clinical samples of urinary nucleic acids possible.

scholarly journals Development of mass spectrometry-based targeted assay for direct detection of novel SARS-CoV-2 coronavirus from clinical specimens

2020 ◽  
Author(s):  
Santosh Renuse ◽  
Patrick M Vanderboom ◽  
Anthony D. Maus ◽  
Jennifer V. Kemp ◽  
Kari M. Gurtner ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution Mass Spectrometry ◽  
Direct Detection ◽  
Diagnostic Testing ◽  
Clinical Samples ◽  
Target Antigen ◽  
Nasopharyngeal Swab ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Ensemble Machine Learning

The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostics including RT-PCR-based assays, antigen detection by lateral flow assays and antibody-based assays have been developed and deployed in a short time. However, many of these assays are lacking in sensitivity and/or specificity. Here, we describe an immunoaffinity purification followed by high resolution mass spectrometry-based targeted assay capable of detecting viral antigen in nasopharyngeal swab samples of SARS-CoV-2 infected individuals. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assays on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was created using fragment ion intensities in the PRM data. This resulted in 97.8% sensitivity and 100% specificity with RT-PCR-based molecular testing as the gold standard. Our results demonstrate that direct detection of infectious agents from clinical samples by mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories.

scholarly journals Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

2020 ◽  
Author(s):  
Piotr Chomczynski
Keyword(s):  
Room Temperature ◽  
Diagnostic Testing ◽  
Viral Rna ◽  
Rt Pcr ◽  
Purification Step ◽  
Biological Specimen ◽  
Rna Detection ◽  
Rna Purification ◽  
Rapid Processing ◽  
Effect Of Inhibitors

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD calculated following U.S. FDA guidelines is 600 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium (UTM), is compatible with heat treatment, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.

scholarly journals Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246867
Author(s):  
Piotr Chomczynski ◽  
Peter W. Chomczynski ◽  
Amy Kennedy ◽  
Michal Rymaszewski ◽  
William W. Wilfinger ◽  
...  
Keyword(s):  
Room Temperature ◽  
Diagnostic Testing ◽  
Viral Rna ◽  
Rt Pcr ◽  
Purification Step ◽  
Biological Specimen ◽  
Rna Detection ◽  
Rna Purification ◽  
Rapid Processing ◽  
Effect Of Inhibitors

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

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