scholarly journals A faster and less costly alternative for RNA extraction of SARS-CoV-2 using proteinase k treatment followed by thermal shock

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248885
Author(s):  
Adolfo Marcelo Ñique ◽  
Fiorella Coronado-Marquina ◽  
Jairo Andrés Mendez Rico ◽  
María Paquita García Mendoza ◽  
Nancy Rojas-Serrano ◽  
...  
Keyword(s):  
Thermal Shock ◽  
Real Time ◽  
Internal Control ◽  
Rna Extraction ◽  
Cost Effective ◽  
Proteinase K ◽  
Rt Pcr ◽  
Critical Material ◽  
Proteinase K Treatment ◽  
Commercial Kit

One of the biggest challenges during the pandemic has been obtaining and maintaining critical material to conduct the increasing demand for molecular tests. Sometimes, the lack of suppliers and the global shortage of these reagents, a consequence of the high demand, make it difficult to detect and diagnose patients with suspected SARS-CoV-2 infection, negatively impacting the control of virus spread. Many alternatives have enabled the continuous processing of samples and have presented a decrease in time and cost. These measures thus allow broad testing of the population and should be ideal for controlling the disease. In this sense, we compared the SARS-CoV-2 molecular detection effectiveness by Real time RT-PCR using two different protocols for RNA extraction. The experiments were conducted in the National Institute of Health (INS) from Peru. We compared Ct values average (experimental triplicate) results from two different targets, a viral and internal control. All samples were extracted in parallel using a commercial kit and our alternative protocol–samples submitted to proteinase K treatment (3 μg/μL, 56°C for 10 minutes) followed by thermal shock (98°C for 5 minutes followed by 4°C for 2 minutes); the agreement between results was 100% in the samples tested. In addition, we compared the COVID-19 positivity between six epidemiological weeks: the initial two in that the Real time RT-PCR reactions were conducted using RNA extracted by commercial kit, followed by two other using RNA obtained by our kit-free method, and the last two using kit once again; they did not differ significantly. We concluded that our in-house method is an easy, fast, and cost-effective alternative method for extracting RNA and conducing molecular diagnosis of COVID-19.

scholarly journals A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation

2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Catia Mio ◽  
Adriana Cifù ◽  
Stefania Marzinotto ◽  
Natascha Bergamin ◽  
Chiara Caldana ◽  
...  
Keyword(s):  
Large Scale ◽  
Rna Extraction ◽  
Cost Effective ◽  
Extraction Methods ◽  
Rapid Identification ◽  
Proteinase K ◽  
Rt Pcr ◽  
Isolation Method ◽  
Pcr Assay ◽  
Sensitivity Specificity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

scholarly journals Validation of saliva sampling as an alternative to oro-nasopharyngeal swab for detection of SARS-CoV-2 using unextracted rRT-PCR with the Allplex 2019-nCoV assay

2021 ◽  
Vol 70 (8) ◽  
Author(s):  
Marco Andres Bergevin ◽  
Wesley Freppel ◽  
Guylaine Robert ◽  
Georges Ambaraghassi ◽  
Dany Aubry ◽  
...  
Keyword(s):  
Detection Efficiency ◽  
Rna Extraction ◽  
Positive Sample ◽  
Proteinase K ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Specialized Equipment ◽  
Potential Alternative ◽  
Proteinase K Treatment ◽  
Alternative Specimen

Introduction. The current severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) pandemic has stressed the global supply chain for specialized equipment, including flocked swabs. Hypothesis. Saliva could be a potential alternative specimen source for diagnosis of SARS-CoV-2 infection by reverse-transcriptase PCR (RT-PCR). Aim. To compare the detection efficiency of SARS-CoV-2 RNA in saliva and oro-nasopharyngeal swab (ONPS) specimens. Methodology. Patients recruited from hospital provided paired saliva and ONPS specimens. We performed manual or automated RT-PCR with prior proteinase K treatment without RNA extraction using the Seegene Allplex 2019 nCoV assay. Results. Of the 773 specimen pairs, 165 (21.3 %) had at least one positive sample. Additionally, 138 specimens tested positive by both sampling methods. Fifteen and 12 cases were detected only by nasopharyngeal swab and saliva, respectively. The sensitivity of ONPS (153/165; 92.7 %; 95 % CI: 88.8–96.7) was similar to that of saliva (150/165; 90.9 %; 95 % CI: 86.5–95.3; P=0.5). In patients with symptoms for ≤ 10 days, the sensitivity of ONPS (118/126; 93.7 %; 95 % CI: 89.4–97.9) was similar to that of saliva (122/126; 96.8 %; 95 % CI: 93.8–99.9 %; P=0.9). However, the sensitivity of ONPS (20/22; 95.2 %; 95 % CI: 86.1–100) was higher than that of saliva (16/22; 71.4 %; 95 % CI: 52.1–90.8) in patients with symptoms for more than 10 days. Conclusions. Saliva sampling is an acceptable alternative to ONPS for diagnosing SARS-CoV-2 infection in symptomatic individuals displaying symptoms for ≤ 10 days. These results reinforce the need to expand the use of saliva samples, which are self-collected and do not require swabs.

scholarly journals Development and validation of cost-effective one-step multiplex RT-PCR assay for detecting the SARS-CoV-2 infection using SYBR Green melting curve analysis

2021 ◽  
Author(s):  
Shovon Lal Sarkar ◽  
A. S. M. Rubayet Ul Alam ◽  
Prosanto Kumar Das ◽  
Md. Hasan Ali Pramanik ◽  
Hassan M. Al-Emran ◽  
...  
Keyword(s):  
Internal Control ◽  
Melting Curve ◽  
Cost Effective ◽  
Curve Analysis ◽  
Sybr Green ◽  
Taqman Probe ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
One Step ◽  
Commercial Kit

TaqMan probe-based expensive commercial real-time (RT) PCR kits are being used in COVID-19 diagnosis. The unprecedented scale of SARS-CoV-2 infections has urgently needed to meet the challenge of testing more persons at a reasonable cost. This study developed a rapid, simple, and cost-effective alternative diagnostic method based on melting curve analysis of SYBR green multiplex assay with a host-specific internal control. A total of 90 randomly selected samples were used for comparing the assay with an available commercial kit to analyse the variation and validity of this in-house developed method. Our customized designed primers specifically detected the virus as similar to commercial kit manufactured by Sansure Biotech Inc. We optimized separately the N, E, S, and RdRp genes by SYBR Green RT-PCR method based on melting curve analysis. Afterwards, a multiplex COVID-19 diagnosis method targeting N and E genes of the virus along with the β-actin gene of the host as an internal control has been established. The total run-time of our proposed method was less than 90 minutes. The cost of each sample processing was less than $2. Overall, this one-step and one-tube method can revolutionize the COVID-19 diagnosis in developing countries.

scholarly journals Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

2021 ◽  
Author(s):  
Rajeev Kumar Jain ◽  
Nagaraj Perumal ◽  
Rakesh Shrivastava ◽  
Kamlesh Kumar Ahirwar ◽  
Jaya Lalwani ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Specific Gene ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Gene Target ◽  
Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

scholarly journals Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR

2005 ◽  
Vol 17 (6) ◽  
pp. 574-578 ◽  
Author(s):  
Ming Y. Deng ◽  
He Wang ◽  
Gordon B. Ward ◽  
Tammy R. Beckham ◽  
Thomas S. McKenna
Keyword(s):  
Real Time ◽  
Classical Swine Fever Virus ◽  
Rna Extraction ◽  
Classical Swine Fever ◽  
Extraction Methods ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcription Pcr ◽  
Rna Extraction Methods ◽  
Positive Results

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription–PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding β-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

SPOT REAL-TIME RT-PCR: A METHOD FOR DIRECT DETECTION OF PLUM POX VIRUS AVOIDING RNA EXTRACTION

2008 ◽  
pp. 215-220
Author(s):  
N. Capote ◽  
E. Bertolini ◽  
M.C. Martínez ◽  
A. Olmos ◽  
M.T. Gorris ◽  
...  
Keyword(s):  
Real Time ◽  
Direct Detection ◽  
Rna Extraction ◽  
Plum Pox Virus ◽  
Rt Pcr

scholarly journals Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

2008 ◽  
Vol 8 (1) ◽  
pp. 131 ◽  
Author(s):  
Marino Expósito-Rodríguez ◽  
Andrés A Borges ◽  
Andrés Borges-Pérez ◽  
José A Pérez
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Development Process ◽  
Rt Pcr ◽  
Internal Control Genes ◽  
Selection Of

scholarly journals Development and Testing of a Method for Detecting Lujo Virus RNA by Reverse Transcription and Real Time Polymerase Chain Reaction

2021 ◽  
pp. 110-115
Author(s):  
E. V. Naidenova ◽  
V. G. Dedkov ◽  
D. A. Agafonov ◽  
A. M. Senichkina ◽  
M. V. Safonova ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Biological Samples ◽  
Control Sample ◽  
Virus Genome ◽  
Positive Control ◽  
Control Panel ◽  
Rt Pcr ◽  
Rna Detection ◽  
Virus Rna

The aim of the study was to develop and assess the efficacy of a method for Lujo virus RNA detection in clinical and biological samples using one-step real-time RT-PCR.Materials and methods. In order to select the conservative regions of the genome, we utilized the available in GenBank database Lujo virus sequences (https://www.ncbi. nlm.nih.gov/genbank) aligned in BioEdit 7.2.5 software package ( (IbisBiosciences, USA). To conduct one-round RTPCR, reverse transcriptase and TaqF-polimerase were used. Recombinant Escherichia coli strain, XL1-Blue, containing pGEM-T plasmid with inserted synthetically-generated fragment of the virus genome, was produced to make positive control sample (PCS). Constructed recombinant plasmids were used for creating RNA-containing PCS with protective protein shell of MS2-phage. Determination of specificity of the developed method was performed with the help of control panel of RNA and DNA of 23 viral strains related to 10 families; the sensitivity – the panel of biological samples artificially contaminated with PCS. Further testing was carried out at the premises of laboratory of the Russian-Guinean Center for Epidemiology and Prevention of Infectious Diseases (Kindia, Republic of Guinea) on 265 blood sera from practically healthy persons, 110 blood sera of cattle, 83 suspensions of ticks, and 165 suspensions of organs of small mammals collected in the territory of Guinea.Results and discussion. Two conservative polymerase gene fragments have been chosen as targets for Lujo virus RNA detection using RT-PCR. The combination of primers and probes has been experimentally selected, optimum composition of reaction mixture for PCR and mode of RT-PCR set-up established, as well as control samples C+, internal control, positive control sample developed. Sensitivity of the proposed method is 5·103  GE /ml, specificity – 100 %. 

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