scholarly journals Utility of Nucleic Acid Extraction Free COVID-19 Real Time PCR Protocol in Resource Limited Setting: A Pilot Study

2021 ◽  
Author(s):  
Santosh Karade ◽  
Pratik Thosani ◽  
Prashant Patil ◽  
Kavita Bala Anand ◽  
Sourav Sen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Pilot Study ◽  
Real Time ◽  
Gold Standard ◽  
Rna Extraction ◽  
Molecular Diagnostic ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Resource Limited

Introduction: Coronavirus Disease (COVID-19), a respiratory infection, caused by severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, Hubei province, China in December 2019. Alarming increase in the number of cases has put tremendous pressure on existing health resources. Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), a molecular diagnostic method, is considered gold standard for diagnosis of SARS-CoV-2 infection. It involves RNA extraction as the preliminary step. Innovations to cut down cost and time involved in SARS-CoV-2 testing are need of hour. Aim: The aim of this study was to assess the feasibility of Nucleic Acid Extraction Free (NEF) protocol for COVID-19 diagnosis in resource limited settings. Materials and Methods: In this pilot study a panel of 148 Nasopharyngeal (NP) samples was subjected to the novel NEF RT-PCR protocol and results were compared to gold standard RT-PCR on RNA extracted from NP specimen. The cycle threshold value for each target was tabulated in MS Excel Spreadsheet and data analysis was performed using Statistical Package for Social Sciences (SPSS) software version 15.0. Results: Out of 148 collected samples, 120 showed amplification of E and RdRp targets by RNA extraction-based RT-PCR. Overall sensitivity and specificity observed for NEF protocol was 43.94% and 96.42%, respectively. Conclusion: Further refinement in the protocol would be required to improve the sensitivity of NEF protocol and widespread use in laboratories.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

scholarly journals Evaluation of real-time nucleic acid sequence-based amplification for detection of Chikungunya virus in clinical samples

2009 ◽  
Vol 58 (9) ◽  
pp. 1168-1172 ◽  
Author(s):  
J.-N. Telles ◽  
K. Le Roux ◽  
P. Grivard ◽  
G. Vernet ◽  
A. Michault
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Chikungunya Virus ◽  
False Negative ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Plasma Samples ◽  
Rt Pcr ◽  
Nucleic Acid Extraction

The Chikungunya virus (CHIKV) is a member of the genus Alphavirus that is transmitted to humans by Aedes mosquitoes. In 2005 and 2006, the Indian Ocean island of La Réunion was hit with an unprecedented CHIKV fever outbreak that infected 300 000 people. In the present study, we describe the evaluation of real-time nucleic acid sequence-based amplification (RT-NASBA) for the detection of CHIKV in clinical samples. A co-extracted and co-amplified chimerical CHIKV RNA sequence was used as an internal control to eliminate false-negative results. The detection threshold of the assay was determined from quantified CHIKV-positive plasma, and estimated to be 200 copies per NASBA reaction. The specificity of the assay was determined using blast analyses and non-cross-reactivity using an O'nyong-nyong virus culture and 250 CHIKV RT-PCR-negative plasma samples. A 100 % specificity was found and no invalid result was obtained, showing the good quality of the nucleic acid extraction. The assay was then evaluated using 252 CHIKV-positive RT-PCR plasma samples. The samples were all tested positive, including those with low viral load. This evaluation showed that the RT-NASBA is a rapid (5 h from sample nucleic acid extraction to detection), sensitive, specific and reliable method for the routine diagnosis of CHIKV in clinical samples.

scholarly journals Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples

2011 ◽  
Vol 77 (13) ◽  
pp. 4336-4343 ◽  
Author(s):  
Akihiko Hata ◽  
Hiroyuki Katayama ◽  
Masaaki Kitajima ◽  
Chettiyappan Visvanathan ◽  
Chea Nol ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Water Samples ◽  
Rna Extraction ◽  
Environmental Water ◽  
Environmental Water Samples ◽  
Acid Extraction ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Viral Nucleic Acid

ABSTRACTInhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Limited-Resource Preparable Chitosan Magnetic Particles for Extracting Amplification-Ready Zeptomole-Range Nucleic Acid from Complex Biofluid

2021 ◽  
Author(s):  
Sayantan Tripathy ◽  
Arunansu Talukdar ◽  
Goutam Pramanik ◽  
P. V. Rajesh ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Magnetic Particles ◽  
Limited Resource ◽  
Nucleic Acid Amplification ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Nucleic Acid Extraction ◽  
Spin Column

<b>Layman Summary: </b>Nucleic acid extraction is a key prerequisite for any nucleic acid amplification test (NAAT) or isothermal NAAT (iNAAT) based molecular diagnosis assays.<b> </b>Existing methods utilizes spin column system for nucleic acid extraction which are unsuitable for limited resource settings. Our work explores two methods for chitosan coated magnetic particle preparation that can be executed within 6 h from commonly available chemicals with nothing but a magnetic stirrer and water bath and doable by a minimally trained person. We will also investigated the compatibility of the extracted nucleic acid with downstream NAATs such as real time LAMP, colorimetric LAMP, and real time PCR. In the process, we established the analytical sensitivity of the overall method.<div><br><div><b>Characterization methods</b>: SEM, XRD, EDX, FT-IR</div><div><br></div><div><b>Bioanalytical methods:</b> Real time LAMP, Colorimetric LAMP, Real time PCR</div></div>

scholarly journals High-throughput HTLV-1 proviral DNA detection system using a nucleic acid extraction robot and real-time PCR detection.

2001 ◽  
Vol 47 (3) ◽  
pp. 378-383 ◽  
Author(s):  
Chieko Matsumoto ◽  
Rieko Shiozawa ◽  
Shigeki Mitsunaga ◽  
Akiko Ichikawa ◽  
Rika Ishiwatari ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Detection System ◽  
Dna Detection ◽  
Pcr Detection ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Proviral Dna

scholarly journals Optimization of Real-time Reverse Transcriptase Polymerase Chain Reaction for Detection of Dengue Virus

2020 ◽  
pp. 1-7
Author(s):  
Kaunara A. Azizi ◽  
Arnold J. Ndaro ◽  
Athanasia Maro ◽  
Adonira Saro ◽  
Reginald A. Kavishe
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Reliable Technique ◽  
Polymerase Chain

Aims: This study was set to optimize conditions for real time reverse transcriptase polymerase chain reaction (RT-PCR) for detection of dengue virus by using rapid and simple nucleic acid extraction method. Methodology: One step and two step real time RT-PCR were evaluated in different PCR thermocyclers. Extraction of viral RNA was done by using a simple boom method. Results: The real time RT-PCR technique was successfully optimized using simple and rapid method for purification of nucleic acid, ‘boom method’. The technique works better when performed in a two-step procedure and can works well with all range of real time PCR machines. The optimized real time RT-PCR used in the present study is a valuable and reliable technique for routine diagnosis of dengue. Further investigation on the cost effectiveness in adopting this technique for routine screening and monitoring of the dengue infection should be done.

scholarly journals Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples

2021 ◽  
Author(s):  
Jennifer R. Hamilton ◽  
Elizabeth C. Stahl ◽  
Connor A. Tsuchida ◽  
Enrique Lin-Shiao ◽  
C. Kimberly Tsui ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rna Extraction ◽  
Detection Sensitivity ◽  
Nasopharyngeal Swab ◽  
Infection Prevalence ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Rapid Pcr ◽  
Large Populations ◽  
Asymptomatic Individuals

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

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