scholarly journals Dry Swab Method of sample collection for SARS-CoV2 testing can be used for culturing virus

2021 ◽  
Author(s):  
Sushma Ram ◽  
M. Ghalib Enayathullah ◽  
Yash Parekh ◽  
Karthik Bharadwaj Tallapaka ◽  
Rakesh K Mishra ◽  
...  
Keyword(s):  
Room Temperature ◽  
Rna Extraction ◽  
Vero Cells ◽  
Sample Collection ◽  
Qpcr Data ◽  
Transport Medium ◽  
Viral Transport ◽  
Viral Particles ◽  
Extraction Step ◽  
Viral Transport Medium

Background: Earlier studies suggested the use of dry swab method for SARS-CoV-2 detection as it does not need VTM and subsequent RNA extraction step making the process cheaper, safer and faster. In this study we explore whether the virus in the dry swab is viable and can be cultured and propagated. Method: Swabs were spiked with SARS-CoV-2 and stored in three different conditions: a) as dry swab (SD, eluted in 1 mL DMEM), b) in 1 mL of Viral Transport Medium (SVTM), and c) in 1 mL of Tris-EDTA buffer (STE). The sample groups were stored either at room temperature (RT ,25°C±1°C) or at 4°C for 1, 4, 8, 12, 24, 48 and 72 hours before being used as viral inoculums for the propagation studies in Vero cells. Results: The RT-qPCR data suggests that SD incubated both at RT and 4°C harbors viral particles that are viable and culturable at par with SVTM and STE. Conclusion: The dry swab method, in addition to its advantages in detection of the virus, also renders viable viral particles that can be cultured and propagated.

scholarly journals Optimal Preparation of COVID-19 Viral Transport Medium for Culture

2020 ◽  
Author(s):  
Julie McAuley ◽  
Claire Fraser ◽  
Elena Paraskeva ◽  
Elizabeth Trajcevska ◽  
Michelle Sait ◽  
...  
Keyword(s):  
Diagnostic Testing ◽  
Vero Cells ◽  
Nucleic Acid Detection ◽  
Transport Medium ◽  
Viral Culture ◽  
Viral Transport ◽  
Viral Transport Medium ◽  
Escape Mutants ◽  
Diagnostic Approaches ◽  
Manual Handling

Abstract IntroductionThe sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained.MethodsVTM was prepared using different methods of sterilization then ‘spiked’ with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR (qPCR).ResultsThe best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4oC, and tested within 48 hours. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM was held at room temperature (RT).DiscussionThe manual handling of VTM production, including filling and sterilization, was optimized. SARS-CoV-2 was spiked into VTM to assess different sterilization methods and measure the effects of storage time and temperature upon VTM performance. While most diagnostic protocols will not require replication competent virus, the use of high quality VTM will allow for the next phase of laboratory analysis in the COVID-19 pandemic, including drug and antibody susceptibility analysis of re-isolated SARS--CoV-2, and for the testing of vaccine escape mutants.

scholarly journals Studi Awal Analisis Molekuler Human Papillomavirus dari Apusan Glans dan Batang Penis

2021 ◽  
Vol 9 (4) ◽  
pp. 433
Author(s):  
Maya Savira ◽  
Resty Yuwandari ◽  
Yossi Maryanti ◽  
Rahmat Azhari Kemal ◽  
Donel S
Keyword(s):  
Human Papillomavirus ◽  
Rna Extraction ◽  
Viral Rna ◽  
Transport Medium ◽  
Viral Transport ◽  
Viral Transport Medium

Pria juga dapat mengalami keganasan akibat infeksi Human Papillomavirus (HPV) serta bertindak sebagai reservoir virus. Metode skrining HPV pada wanita telah terstandardisasi, namun belum ada standar metode skrining pada pria di Indonesia. Beberapa studi pada populasi pria di luar negeri menunjukkan potensi sampling pada daerah genitalia eksterna untuk skrining HPV. Tujuan: mengoptimasi metode skrining HPV secara molekuler pada pria. Metode: Responden adalah partner seksual wanita pansien kanker serviks di RSUD Arifin Achmad Provinsi Riau. Apusan dari glans dan batang penis diambil menggunakan nylon-flocked swab yang kemudian dimasukkan ke dalam 350µl viral transport medium terpisah. DNA diisolasi dari sampel yang kemudian dianalisis untuk mendeteksi gen human β-globin dan HPV. Hasil: Optimasi awal menunjukkan gen β-globin dapat terdeteksi dari hasil ekstraksi dengan kit Zeesan Viral RNA Extraction. Pita HPV hasil PCR dengan primer MY09 dan MY11 dapat muncul namun masih tipis. Simpulan: Studi awal ini menunjukkan bahwa apusan glans dan batang penis dapat digunakan untuk deteksi HPV secara molekuler pada pria, namun proses pengambilan sampel, ekstraksi DNA, dan PCR masih perlu dioptimasi.Kata kunci: apusan, glans, HPV, penis

scholarly journals SARS-CoV-2 detection in setting of viral swab scarcity: are MRSA swabs and viral swabs equivalent?

2020 ◽  
Author(s):  
Daniel G. Federman ◽  
Shaili Gupta ◽  
Gary Stack ◽  
Sheldon M. Campbell ◽  
David R. Peaper ◽  
...  
Keyword(s):  
Nasal Swab ◽  
High Rate ◽  
Test Results ◽  
Sample Collection ◽  
Transport Medium ◽  
Viral Transport ◽  
Global Pandemic ◽  
Viral Transport Medium ◽  
Nasal Swabs ◽  
Viral Media

AbstractBackgroundThe global pandemic of Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV2) has resulted in unprecedented challenges for healthcare systems. One barrier to widespread testing has been a paucity of traditional respiratory viral swab collection kits relative to the demand. Whether other sample collection kits, such as widely available MRSA nasal swabs can be used to detect SARS-CoV-2 is unknown.MethodsWe compared simultaneous nasal MRSA swabs (COPAN ESwabs ® 480C flocked nasal swab in 1mL of liquid Amies medium) and virals wabs (BD H192(07) flexible mini-tip flocked nasopharyngeal swabs in 3mL Universal Transport Medium) for SARS-CoV-2 PCR testing using Simplexa COVID-19 Direct assay on patients over a 4-day period. When the results were discordant, the viral swab sample was run again on the Cepheid Xpert Xpress ® SARS-CoV-2 assay.ResultsOf the 81 included samples, there were 19 positives and 62 negatives in viral media and 18 positives and 63 negative in the MRSA swabs. Amongst all included samples, there was concordance between the COPAN ESwabs ® 480C and the viral swabs in 78 (96.3%).ConclusionWe found a high rate of concordance in test results between COPAN ESwabs ® 480C in Amies solution and BD H192(07) nasopharyngeal swabs in in 3 mL of Universal Viral Transport medium viral media. Clinicians and laboratories should feel better informed and assured using COPAN ESwabs ® 480C to help in the diagnosis of COVID-19.

scholarly journals Optimal preparation of SARS-CoV-2 viral transport medium for culture

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Julie McAuley ◽  
Claire Fraser ◽  
Elena Paraskeva ◽  
Elizabeth Trajcevska ◽  
Michelle Sait ◽  
...  
Keyword(s):  
Diagnostic Testing ◽  
Vero Cells ◽  
Nucleic Acid Detection ◽  
Transport Medium ◽  
Viral Culture ◽  
Viral Transport ◽  
Viral Transport Medium ◽  
Escape Mutants ◽  
Diagnostic Approaches ◽  
Manual Handling

Abstract Introduction The sudden arrival of the COVID-19 pandemic placed significant stresses on supply chains including viral transport medium (VTM). The VTM that was urgently required needed to support viral replication, as well as other routine diagnostic approaches. We describe the preparation and validation testing of VTM for rapidly expanding diagnostic testing, where the capacity of the VTM to preserve viral integrity, for culture, isolation and full sequence analysis, was maintained. Methods VTM was prepared using different methods of sterilization then ‘spiked’ with virus. The VTM was investigated using viral culture in Vero cells, and for nucleic acid detection by quantitative PCR. Results The best results were obtained by filter and autoclave-based sterilization. The VTM proved robust for culture-based analyses provided the inoculated VTM was stored at 4 °C, and tested within 48 h. The filtered VTM also supported PCR-based diagnosis for at least 5 days when the mock inoculated VTM was held at room temperature. Discussion The manual handling of VTM production, including filling and sterilization, was optimized. SARS-CoV-2 was spiked into VTM to assess different sterilization methods and measure the effects of storage time and temperature upon VTM performance. While most diagnostic protocols will not require replication competent virus, the use of high quality VTM will allow for the next phase of laboratory analysis in the COVID-19 pandemic, including drug and antibody susceptibility analysis of re-isolated SARS-CoV-2, and for the testing of vaccine escape mutants.

scholarly journals Extraction Free Rapid Detection of SARS-CoV-2 from Oropharyngeal/Nasopharyngeal Swabs by Real Time PCR

2021 ◽  
Author(s):  
Parul Sinha ◽  
Sandeep Gupta ◽  
Megha Gupta ◽  
Dinesh Kumar Jain ◽  
Malvika Sharma ◽  
...  
Keyword(s):  
Real Time ◽  
Extraction Method ◽  
Rna Extraction ◽  
Molecular Testing ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Transport Medium ◽  
Viral Transport ◽  
Viral Transport Medium ◽  
Medium Sample

Introduction: The emergence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has been troublesome particularly for developing countries that lack infrastructure and capacities to produce the kits locally. Simplification of the method can increase diagnostic efficiency which can benefit patients and help in infection control, consequently saving time and lives. Aim: To evaluate the diagnostic value of four methods (that omit extraction step) for detection of SARS-CoV-2 against the traditional extraction method. Materials and Methods: This was a cross-sectional analysis for evaluating diagnostic accuracy of four methods for detection of SARS-CoV-2 by real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR), conducted in the Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India, in October 2020. Ninety four SARS-CoV-2 RT-PCR positive samples and 20 negative samples were taken for this study. Automated extraction system was used for Ribonucleic Acid (RNA) extraction and four different approaches were compared to the traditional extraction method for detection of SARS-CoV-2 by RT-PCR. Data was entered and analysed using Statistical Package for the Social Sciences (SPSS) statistical software version 24.0. Results: The automated RNA extraction method was compared to the method of direct addition of samples with (Heat processed Direct Viral transport medium Sample (HDVS)) and without heating (Direct Viral transport medium Sample (DVS)), directs addition of diluted (1:5) sample with (Heat processed diluted VTM sample (HdVS)) and without heating (Diluted VTM sample (dVS)) as well as after addition of Proteinse K (PK) to the diluted samples that came either negative/invalid. Out of four methods, the HdVS method gave the best results, considering extraction with Perkin Elmer as standard, this method showed sensitivity of 96.74%, specificity of 100%. Conclusion: In current pandemic, molecular testing is critically challenged by the limited supplies of reagents of nucleic acid extraction alternative method like diluting and heating of Viral Transport Media (VTM) samples and using them directly as elutes serve as an easy, fast and inexpensive alternative.

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

2020 ◽  
Vol 15 (15) ◽  
pp. 1483-1487
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Kimya Jones ◽  
...  
Keyword(s):  
Supply Chain ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Short Supply ◽  
Viral Transport ◽  
3D Printed ◽  
Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

scholarly journals Self-collected gargle specimen as a patient-friendly sample collection method for COVID-19 diagnosis in a population context

2021 ◽  
Author(s):  
Revata Utama ◽  
Rebriarina Hapsari ◽  
Iva Puspitasari ◽  
Desvita Sari ◽  
Meita Hendrianingtyas ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Room Temperature ◽  
Rna Extraction ◽  
User Preference ◽  
Sample Collection ◽  
Rt Pcr ◽  
Collection Method ◽  
Qrt Pcr ◽  
Alternative Specimen ◽  
Temperature Storage

Abstract Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.

scholarly journals Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2

2020 ◽  
Author(s):  
A. Ganguli ◽  
A. Mostafa ◽  
J. Berger ◽  
M. Aydin ◽  
F. Sun ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Point Of Care ◽  
Rna Extraction ◽  
Clinical Diagnostics ◽  
Isothermal Amplification ◽  
Sample Collection ◽  
Rt Pcr ◽  
Point Of Use ◽  
Viral Transport Medium

AbstractThe COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/μL in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/μL in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests.Significance StatementAn important limitation of the current assays for the detection of SARS-CoV-2 stem from their reliance on time- and labor-intensive and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative portable platforms that can provide rapid and accurate diagnosis, potentially at the point-of-use. Here, we present the details of an isothermal amplification-based detection of SARS-CoV-2, including the demonstration of a smartphone-based point-of-care device that can be used at the point of sample collection.

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