A two-pronged approach for rapid and high-throughput SARS-CoV-2 nucleic acid testing

AbstractImproved molecular screening and diagnostic tools are needed to substantially increase SARS-CoV-2 testing capacity and throughput while reducing the time to receive test results. Here we developed multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) for detection of SARS-CoV-2 using rapid DNA electrophoresis and alternatively using multiplex viral sequencing (mVseq). For RNA specimens extracted from nasopharyngeal (NP) swabs in viral transport media (VTM), our assays achieved a sensitivity for SARS-CoV-2 detection corresponding to cycle threshold (Ct) of 37.2 based on testing of these specimens using quantitative reverse transcription PCR (RT-qPCR). For NP swab-VTM specimens without prior RNA extraction, sensitivity was reduced to Ct of 31.6, which was due to lower concentration of SARS-CoV-2 genome copies in VTM compared to RNA-extracted samples. Assay turnaround time was 60 minutes using rapid gel electrophoresis, 90 minutes using Agilent Bioanalyzer, and 24-48 hours using Illumina sequencing, the latter of which required a second PCR to produce a sequence-ready library using m-RT-PCR products as the template. Our assays can be employed for high-throughput sequencing-based detection of SARS-CoV-2 directly from a clinical specimen without RNA isolation, while ease-of-use and low cost of the electrophoresis-based readout enables screening, particularly in resource-constrained settings.

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Direct-RT-qPCR Detection of SARS-CoV-2 without RNA Extraction as Part of a COVID-19 Testing Strategy: From Sample to Result in One Hour

Diagnostics ◽

10.3390/diagnostics10080605 ◽

2020 ◽

Vol 10 (8) ◽

pp. 605 ◽

Cited By ~ 3

Author(s):

Eva Kriegova ◽

Regina Fillerova ◽

Petr Kvapil

Keyword(s):

High Throughput Screening ◽

High Speed ◽

Limit Of Detection ◽

Point Of Care ◽

Rna Extraction ◽

Rna Isolation ◽

High Sensitivity ◽

Ease Of Use ◽

Diagnostic Tools ◽

Heat Inactivation

Due to the lack of protective immunity in the general population and the absence of effective antivirals and vaccines, the Coronavirus disease 2019 (COVID-19) pandemic continues in some countries, with local epicentres emerging in others. Due to the great demand for effective COVID-19 testing programmes to control the spread of the disease, we have suggested such a testing programme that includes a rapid RT-qPCR approach without RNA extraction. The Direct-One-Step-RT-qPCR (DIOS-RT-qPCR) assay detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one hour while maintaining the high sensitivity and specificity required of diagnostic tools. This optimised protocol allows for the direct use of swab transfer media (14 μL) without the need for RNA extraction, achieving comparable sensitivity to the standard method that requires the time-consuming and costly step of RNA isolation. The limit of detection for DIOS-RT-qPCR was lower than seven copies/reaction, which translates to 550 virus copies/mL of swab. The speed, ease of use and low price of this assay make it suitable for high-throughput screening programmes. The use of fast enzymes allows RT-qPCR to be performed under standard laboratory conditions within one hour, making it a potential point-of-care solution on high-speed cycling instruments. This protocol also implements the heat inactivation of SARS-CoV-2 (75 °C for 10 min), which renders samples non-infectious, enabling testing in BSL-2 facilities. Moreover, we discuss the critical steps involved in developing tests for the rapid detection of COVID-19. Implementing rapid, easy, cost-effective methods can help control the worldwide spread of the COVID-19 infection.

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A simple and rapid method for isolating high-quality RNA from kenaf containing high polysaccharide and polyphenol contents

10.1101/2020.07.06.189506 ◽

2020 ◽

Author(s):

Xiaofang Liao ◽

Hongwei Li ◽

Aziz Khan ◽

Yanhong Zhao ◽

Wenhuan Hou ◽

...

Keyword(s):

Low Cost ◽

Rna Extraction ◽

Rna Isolation ◽

Cost Effective ◽

Spectrophotometric Analysis ◽

Rt Pcr ◽

High Quality ◽

Time Saving ◽

Ctab Method ◽

Commercial Kits

AbstractThe isolation of high-quality RNA from kenaf is crucial for genetic and molecular biology studies. However, high levels of polysaccharide and polyphenol compounds in kenaf tissues could irreversibly bind to and coprecipitate with RNA, which complicates RNA extraction. In the present study, we proposed a simplified, time-saving and low-cost extraction method for isolating high quantities of high-quality RNA from several different kenaf tissues. RNA quality was measured for yield and purity, and the proposed protocol yielded high quantities of RNA (10.1-12.9 μg/g·FW). Spectrophotometric analysis showed that A260/280 ratios of RNA samples were in the range of 2.11 to 2.13, and A260/230 ratios were in the range of 2.04-2.24, indicating that the RNA samples were free of polyphenols, polysaccharides, and protein contaminants after isolation. The method of RNA extraction presented here was superior to the conventional CTAB method in terms of RNA isolation efficiency and was more sample-adaptable and cost-effective than commercial kits. Furthermore, to confirm downstream amenability, the high-quality RNA obtained from this method was successfully used for RT-PCR, real-time RT-PCR and Northern blot analysis. We provide an efficient and low-cost method for extracting high quantities of high-quality RNA from plants that are rich in polyphenols and polysaccharides, and this method was also validated for the isolation of high-quality RNA from other plants.

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In-House, Rapid, and Low-Cost SARS-CoV-2 Spike Gene Sequencing Protocol to Identify Variants of Concern Using Sanger Sequencing

10.1101/2021.08.09.21261723 ◽

2021 ◽

Author(s):

Fatimah Alhamlan ◽

Dana Bakheet ◽

Marie Bohol ◽

Madain Alsanea ◽

Basma Alahaideb ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

High Throughput ◽

Sanger Sequencing ◽

High Throughput Sequencing ◽

Low Cost ◽

Rna Extraction ◽

Whole Genome ◽

Diagnostic Laboratory ◽

Spike Gene

Background: The need for active genomic sequencing surveillance to rapidly identify circulating SARS-CoV-2 variants of concern (VOCs) is critical. However, increased global demand has led to a shortage of commercial SARS-CoV-2 sequencing kits, and not every country has the technological capability or the funds for high-throughput sequencing platforms. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol that uses supplies, equipment, and methodologic expertise available in standard molecular or diagnostic laboratories to identify circulating SARS-CoV-2 variants of concern. Methods: Sets of primers flanking the SARS-CoV-2 spike gene were designed using SARS-CoV-2 genome sequences retrieved from the Global Initiative on Sharing Avian Influenza Data (GISAID) Database and synthesized in-house. Primer specificity and final sequences were verified using online prediction analyses with BLAST. The primers were validated using 282 nasopharyngeal samples collected from patients assessed as positive for SARS-CoV-2 at the diagnostic laboratory of the hospital using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit. The patient samples were subjected to RNA extraction followed by cDNA synthesis, conventional polymerase chain reaction, and Sanger sequencing. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole genome sequencing of the same samples. Results: Sanger sequencing using the newly designed primers and next-generation whole genome sequencing of 282 patient samples indicated identical variants of concern results: 123 samples contained the alpha variant (B.1.1.7); 78, beta (B.1.351), 0, gamma (P.1), and 13, delta (B.1.617.2). Moreover, the remaining samples were non-VOC that belonged to none of these variants and had 99.97% identity with the reference genome. Only four samples had poor sequence quality by Sanger sequencing owing to a low viral count (Ct value >38). Therefore, mutation calls were >98% accurate. Conclusions: Sanger sequencing method using in-house primers is an alternative approach that can be used in facilities with existing equipment to mitigate limitations in high throughput supplies required to identify SARS-CoV-2 variants of concern during the COVID-19 pandemic. This protocol is easily adaptable for detection of emerging variants.

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

Viruses ◽

10.3390/v13040615 ◽

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.

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Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids

Virology Journal ◽

10.1186/s12985-021-01523-1 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Rachelle Bester ◽

Glynnis Cook ◽

Johannes H. J. Breytenbach ◽

Chanel Steyn ◽

Rochelle De Bruyn ◽

...

Keyword(s):

High Throughput ◽

Extraction Method ◽

Pathogen Detection ◽

High Throughput Sequencing ◽

Expression Profiles ◽

Rna Extraction ◽

Healthy Plant ◽

Ion Torrent ◽

Extraction Protocol ◽

Zymo Research

Abstract Background High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

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Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽

10.1094/pdis-05-21-0949-re ◽

2021 ◽

Author(s):

Dan Edward Veloso Villamor ◽

Karen E Keller ◽

Robert Martin ◽

Ioannis Emmanouil Tzanetakis

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Wide Spectrum ◽

Virus Detection ◽

Rt Pcr ◽

Host Interactions ◽

Growing Seasons ◽

Berry Crops ◽

Sampling Times ◽

Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

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Efficient High-throughput Techniques for the Analysis of Disease-Resistant Plant Varieties and Detection of Food Adulteration

Current Protein and Peptide Science ◽

10.2174/1389203723666211223111238 ◽

2021 ◽

Vol 23 ◽

Author(s):

Romesh Kumar Salgotra ◽

Rafiq Ahmad Bhat ◽

Deyue Yu ◽

Javaid Akhter Bhat

Keyword(s):

Multiplex Pcr ◽

High Throughput ◽

High Throughput Sequencing ◽

Low Cost ◽

Crop Improvement ◽

Small Sample ◽

Food Crops ◽

Genomic Resources ◽

Breeding Programmes ◽

Next Generation Sequencing Ngs

Abstract: Over the past two decades, the advances in the next generation sequencing (NGS) platforms have led to the identification of numerous genes/QTLs at high-resolution for their potential use in crop improvement. The genomic resources generated through these high-throughput sequencing techniques have been efficiently used in screening of particular gene of interest particularly for numerous types of plant stresses and quality traits. Subsequently, the identified-markers linked to a particular trait have been used in marker-assisted backcross breeding (MABB) activities. Besides, these markers are also being used to catalogue the food crops for detection of adulteration to improve the quality of food. With the advancement of technologies, the genomic resources are originating with new markers; however, to use these markers efficiently in crop breeding, high-throughput techniques (HTT) such as multiplex PCR and capillary electrophoresis (CE) can be exploited. Robustness, ease of operation, good reproducibility and low cost are the main advantages of multiplex PCR and CE. The CE is capable of separating and characterizing proteins with simplicity, speed and small sample requirements. Keeping in view the availability of vast data generated through NGS techniques and development of numerous markers, there is a need to use these resources efficiently in crop improvement programmes. In summary, this review describes the use of molecular markers in the screening of resistance genes in breeding programmes and detection of adulterations in food crops using high-throughput techniques.

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Novel Approaches to Diagnose COVID-19

Kurdistan Journal of Applied Research ◽

10.24017/covid.7 ◽

2020 ◽

pp. 66-71

Author(s):

Solaf Jawhar Ali

Keyword(s):

Clinical Symptoms ◽

Low Cost ◽

Case Definition ◽

Diagnostic Tools ◽

Rt Pcr ◽

Public Health Agencies ◽

Health Agencies ◽

Acute Respiratory Diseases ◽

Diagnostic Approaches ◽

Laboratory Examinations

An widespread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in December 2019 has occurred worldwide. Public health agencies are in need of developing diagnostic tools which will have a major impact in tracking the virus and suppressing the transmission. Diagnosis of the disease is based on clinical symptoms, epidemiological history and laboratory examinations. Severe acute respiratory diseases with fever and ,cough and dyspnea, are used as the case definition to select people for testing. Different samples taken from the human body such as oropharyngeal (OP) and nasopharyngeal (NP) swabs are used to detect the virus. SARS-CoV-2 can be detected with different methods in the laboratory including real time RT-PCR, chest CT scan and immunoassays. Viral nucleic acid testing has played important role in control COVIDI-19 outbreak. More recently, a new CRISPR-based DETECTR assay has been developed to detect COVID-19. This test is rapid (~30 min), low-cost, and precise for identification of SARS-CoV-2. In addition, immunoassays and medical imaging can use as supplementary tests, combined with RT-PCR. This review is conducted to summarizes the current information on the present diagnostic approaches for SARS-CoV-2.

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DNA metabarcoding of insects and allies: an evaluation of primers and pipelines

Bulletin of Entomological Research ◽

10.1017/s0007485315000681 ◽

2015 ◽

Vol 105 (6) ◽

pp. 717-727 ◽

Cited By ~ 83

Author(s):

G.-J. Brandon-Mong ◽

H.-M. Gan ◽

K.-W. Sing ◽

P.-S. Lee ◽

P.-E. Lim ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Illumina Miseq ◽

Ease Of Use ◽

Malaise Trap ◽

Detection Rates ◽

Operational Taxonomic Units ◽

Arthropod Species ◽

Primer Sets ◽

Arthropod Biodiversity

AbstractMetabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80–90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

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