Optimization of multiplex PCR composition to screen for SARS-CoV-2 variants of concern

2021 ◽  
Vol 4 (2) ◽  
pp. 58
Author(s):  
Maya Savira ◽  
Enikarmila Asni ◽  
Rahmat Azhari Kemal
Keyword(s):  
Whole Genome Sequencing ◽  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Screening Method ◽  
Positive Control ◽  
Whole Genome ◽  
Rt Pcr ◽  
Pcr Screening ◽  
Single Target ◽  
Alpha Variant

Background: The ongoing COVID-19 pandemic has led to the emergence of several variants of concern. To rapidly identify those variants, screening samples for whole-genome sequencing (WGS) prioritization could be performed.  Objective: We optimized the polymerase chain reaction (PCR) screening method to identify the mutation in spike and ORF1a regions.  Methods: We adopted primers targeting mutation in spike and ORF1a region from another study. We optimized the PCR screening method using kits readily available in Indonesia. Firstly, we compared N1 and N2 primers as internal positive control. We also compared GoTaq® 1-Step RT-qPCR System and Indonesia TFRIC-19 BioCOV-19 for the multiplex reaction. We used the optimized composition to screen SARS-CoV-2 positive samples from April – June 2021. Samples with spike and/or ORF1a target failure were subjected to whole genome sequencing (WGS).  Results: The results demonstrated the N2 BioCOV-19 reaction as the optimized multiplex PCR composition for spike and ORF1a mutations screening. Whole-genome sequencing has shown that a sample with spike and ORF1a targets failure to be Alpha variant, while other samples with single target failure as non-variants of concern. Therefore, a multiplex RT-PCR composition has been optimized to detect mutation in spike and ORF1a regions. Conclusion: We have optimized a multiplex RT-PCR composition to detect mutation in spike and ORF1a regions.

scholarly journals Severe SARS-CoV-2 Breakthrough Reinfection With Delta Variant After Recovery From Breakthrough Infection by Alpha Variant in a Fully Vaccinated Health Worker

2021 ◽  
Vol 8 ◽  
Author(s):  
Jayanthi Shastri ◽  
Swapneil Parikh ◽  
Veena Aggarwal ◽  
Sachee Agrawal ◽  
Nirjhar Chatterjee ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Index Case ◽  
Whole Genome ◽  
Chest Imaging ◽  
Rt Pcr ◽  
Breakthrough Infection ◽  
Acute Phase Reactants ◽  
Post Vaccination ◽  
Alpha Variant

Background: Post infection immunity and post vaccination immunity both confer protection against COVID-19. However, there have been many whole genome sequencing proven reinfections and breakthrough infections. Both are most often mild and caused by Variants of Concern (VOC).Methods: The patient in our study underwent serial COVID-19 RT-PCR, blood tests for serology, acute phase reactants, and chest imaging as part of clinical care. We interviewed the patient for clinical history and retrieved reports and case papers. We retrieved stored RT-PCR positive samples for whole genome sequencing (WGS) of SARS-CoV-2 from the patient's breakthrough infections and the presumed index case.Findings: The patient had three RT-PCR confirmed SARS-CoV-2 infections. Two breakthrough infections occurred in quick succession with the first over 3 weeks after complete vaccination with COVISHIELD and despite post-vaccination seroconversion. The first breakthrough infection was due to the Alpha variant and the second due to the Delta variant. The Delta variant infection resulted in hypoxia, hospitalization, and illness lasting seven weeks. Serial serology, acute phase reactants, and chest imaging supported WGS in establishing distinct episodes of infection. WGS established a fully vaccinated family member as the index case.Interpretation: The patient had an Alpha variant breakthrough infection despite past infection, complete vaccination, and seroconversion. Despite boosting after this infection, the patient subsequently had a severe Delta variant breakthrough infection. This was also a WGS proven reinfection and, therefore, a case of breakthrough reinfection. The patient acquired the infection from a fully vaccinated family member.

scholarly journals False COVID-19 cases due to contamination by inactivated virus vaccine

2021 ◽  
Author(s):  
Kelvin Kai-Wang To ◽  
Xin Li ◽  
David Christopher Lung ◽  
Jonathan Daniel Ip ◽  
Wan-Mui Chan ◽  
...  
Keyword(s):  
Public Health ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Virus Vaccine ◽  
False Positive ◽  
Spike Protein ◽  
Whole Genome ◽  
Rt Pcr ◽  
Health Measures ◽  
Respiratory Specimens

Abstract A false-positive SARS-CoV-2 RT-PCR result can lead to unnecessary public-health measures. We report two individuals whose respiratory specimens were contaminated by inactivated SARS-CoV-2 vaccine strain(CoronaVac), likely at vaccination premises. Incidentally, whole-genome sequencing of CoronaVac showed adaptive deletions on the spike protein, which do not result in observable changes of antigenicity.

scholarly journals Detection of SARS-CoV-2 from Patient Fecal Samples by Whole Genome Sequencing

2020 ◽  
Author(s):  
Andreas Papoutsis ◽  
Thomas Borody ◽  
Siba Dolai ◽  
Jordan Daniels ◽  
Skylar Steinberg ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genome Analysis ◽  
Nasopharyngeal Swab ◽  
Whole Genome ◽  
Rt Pcr ◽  
Whole Genome Analysis ◽  
Fecal Samples ◽  
Oral Transmission ◽  
Study Participants

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment NGS from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal-oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication.

scholarly journals Identification of nsp1 gene as the target of SARS‐CoV‐2 real‐time RT‐PCR using nanopore whole‐genome sequencing

2020 ◽  
Vol 92 (11) ◽  
pp. 2725-2734 ◽  
Author(s):  
Wan‐Mui Chan ◽  
Jonathan Daniel Ip ◽  
Allen Wing‐Ho Chu ◽  
Cyril Chik‐Yan Yip ◽  
Lap‐Sum Lo ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Rt Pcr ◽  
Nsp1 Gene

scholarly journals A Case of IMP-4-, OXA-421-, OXA-96-, and CARB-2-Producing Acinetobacter pittii Sequence Type 119 in Australia

2014 ◽  
Vol 53 (2) ◽  
pp. 727-730 ◽  
Author(s):  
Witchuda Kamolvit ◽  
Petra Derrington ◽  
David L. Paterson ◽  
Hanna E. Sidjabat
Keyword(s):  
Whole Genome Sequencing ◽  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Draft Genome ◽  
Sequence Type ◽  
Species Level ◽  
Whole Genome ◽  
Patient Identification ◽  
Content Type ◽  
Acinetobacter Pittii

An IMP-4-producingAcinetobacterpittiistrain coproducing oxacillinases was isolated from a leg wound of a 67-year-old female patient. Identification to the species level byrpoBandgyrBsequencing and multiplex-PCR-based analysis revealed that the isolate wasA. pittii. Whole-genome sequencing of thisA. pittiiisolate determined the presence ofblaOXA-96,blaCARB-2, and a novelblaOXA-421gene. The position of this novelblaOXA-421gene was similar to that ofblaOXA-51inA. baumannii, downstream of the phosphinothricinN-acetyltransferase gene and upstream offxsAin the chromosome. ThisA. pittiiisolate was found to belong to sequence type 119 (ST119). Here, we report the first isolation of IMP-4-producingA. pittiiST119 with a novelblaOXA-421gene from a patient in Australia and characterize its draft genome.

scholarly journals Genomic diversity of SARS-CoV-2 in Pakistan during fourth wave of pandemic

2021 ◽  
Author(s):  
Massab Umair ◽  
Aamer Ikram ◽  
Zaira Rehman ◽  
Adnan Haider ◽  
Nazish Badar ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genomic Diversity ◽  
Whole Genome ◽  
Mutation Profile ◽  
Alpha Variant ◽  
Fourth Wave

AbstractThe emergence of different variants of concern of SARS-CoV-2 has resulted in upsurges of COVID positive cases around the globe. Pakistan is also experiencing fourth wave of COVID-19 with increasing number of positive cases. In order to understand the genomic diversity of circulating SARS-CoV-2 strains during fourth wave of pandemic in Pakistan, the current study was designed. The samples from 89 COVID-19 positive patients were subjected to whole genome sequencing using GeneStudio S5. The results showed that 99% (n=88) of isolates belonged to delta variant and only one isolate belonged to alpha variant. Among delta variant cases 26.1% (n=23) isolates were showing B.1.617.2 while 74% of isolates showing AY.4 lineage. Islamabad was found to be the most affected city with 54% (n=48) of cases, followed by Karachi (28%, n=25), and Rawalpindi (10%, n=9). AY.4 has slight difference in mutation profile compared to B.1.617.2. E156del, G142D and V26I mutations in spike and T181I in NSP6 were present in B.1.617.2 but not in AY.4. Interestingly, A446V mutation in NSP4 has been only observed in AY.4. The current study highlights the circulation of primarily delta variant (B.1.617.2 and AY.4) during fourth wave of pandemic in Pakistan.

scholarly journals Assessment of Ion S5 NGS protocol for SARS-CoV-2 genome sequencing

2021 ◽  
Vol 5 (2) ◽  
pp. 24
Author(s):  
Dino Pećar ◽  
Ivana Čeko ◽  
Lana Salihefendić ◽  
Rijad Konjhodžić
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Magnetic Beads ◽  
Rna Isolation ◽  
Whole Genome ◽  
Library Preparation ◽  
Rt Pcr ◽  
Sequence Coverage ◽  
Cdna Synthesis ◽  
Good Laboratory

Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.

scholarly journals Emergence of SARS-CoV-2 variant B.1.575.2 containing the E484K mutation in the spike protein in Pamplona (Spain) May-June 2021

2021 ◽  
Author(s):  
Camino Trobajo-Sanmartín ◽  
Ana Miqueleiz ◽  
María Eugenia Portillo ◽  
Miguel Fernández-Huerta ◽  
Ana Navascués ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Threshold Value ◽  
Spike Protein ◽  
Whole Genome ◽  
Viral Dynamics ◽  
Rt Pcr ◽  
Northern Spain ◽  
Novel Mutations ◽  
Rapid Control

With the emergence of new SARS-CoV-2 variants and the acquisition of novel mutations in exiting lineages, the need to implement methods capable of monitoring viral dynamics arises. We report the emergence and spread of a new SARS-CoV-2 variant within B.1.575 lineage containing the E484K mutation in the spike protein (named B.1.575.2) in a region of Northern Spain between May and June 2021. SARS-CoV-2 positive samples with cycle threshold value less than or equal to 30 were selected to screen of presumptive variants using the TaqPathTM COVID-19 RT-PCR kit and TaqManTM SARS-CoV-2 Mutation Panel. Confirmation of variants was performed by whole genome sequencing. Of the 200 samples belonging to the B.1.575 lineage, 194 (97%) corresponded to the B.1.575.2 sub-lineage, which was related to the presence of the E484K mutation. Of 197 cases registered in GISAID EpiCoV database as lineage B.1.575.2 194 (99.5%) were identified in Pamplona (Spain). This report emphasizes the importance of complementing surveillance of SARS-CoV-2 with sequencing for the rapid control of emerging viral variants.

scholarly journals Serotyping of sub-Saharan Africa Salmonella Strains Isolated from Different Sources Using Multiplex PCR and Capillary Electrophoresis Analysis and whole Genome Sequencing

2020 ◽  
Author(s):  
Assèta Kagambèga ◽  
Lari M. Hiott ◽  
David S. Boyle ◽  
Elizabeth A. McMillan ◽  
Kaisa Haukka ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Whole Genome Sequencing ◽  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Sub Saharan Africa ◽  
Whole Genome ◽  
Sub Saharan ◽  
Different Sources

Abstract The authors have withdrawn this preprint due to author disagreement.

scholarly journals Viral loads and profile of the patients infected with SARS-CoV-2 Delta, Alpha or R.1 variants in Tokyo

2021 ◽  
Author(s):  
Chihiro Tani-Sassa ◽  
Yumi Iwasaki ◽  
Naoya Ichimura ◽  
Katsutoshi Nagano ◽  
Yuna Takatsuki ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Whole Genome ◽  
Elderly Individuals ◽  
Viral Loads ◽  
Copy Numbers ◽  
Significant Difference ◽  
Rapid Spread ◽  
Alpha Variant

The rapid spread of the Delta variant of SARS-CoV-2 became a serious concern worldwide in summer 2021. We examined the copy number and variant types of all SARS-CoV-2-positive patients who visited our hospital from February to August 2021 using PCR tests. Whole genome sequencing was performed for some samples. The R.1 variant (B.1.1.316) was responsible for most infections in March, replacing the previous variant (B.1.1.214); the Alpha (B.1.1.7) variant caused most infections in April and May; and the Delta variant (B.1.617.2) was the most prevalent in July and August. There was no significant difference in copy numbers among the previous variant cases (n=29, median 3.0x104 copies/μL), R.1 variant cases (n=28, 2.1x105 copies/μL), Alpha variant cases (n=125, 4.1x105 copies/μL), and Delta variant cases (n=106, 2.4x105 copies/μL). Patients with Delta variant infection were significantly younger than those infected with R.1 and the previous variants, possibly because many elderly individuals in Tokyo were vaccinated between May and August. There was no significant difference in mortality among the four groups. Our results suggest that the increased infectivity of Delta variant may be caused by factors other than the higher viral loads. Clarifying these factors is important to control the spread of Delta variant infection.

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