NEBNext® Varskip Short ARTIC SARS-CoV-2 RT-PCR Module E7626 v1

2021 ◽  
Author(s):  
New England not provided Biolabs
Keyword(s):  
Rt Pcr ◽  
Cdna Synthesis ◽  
Amplification Reaction

This protocol details methods for the NEBNext® ARTIC SARS-CoV-2 RT-PCR Module, NEB #E7626S/L 24/96 reactions. cDNA Synthesis and Targeted cDNA Amplification with NEBNext VarSkip Short Primer Mixes: This protocol follows an alternate variant-tolerant approach for targeting SARS-CoV-2 by utilizing NEBNext VarSkip Short SARS-CoV-2 Primer Mixes. The NEBNext VarSkip Short SARS-CoV-2 Primer mixes cannot be added to the same cDNA amplification reaction as the NEBNext ARTIC SARS-CoV-2 Primer Mixes. If downstream applications include sequencing, performing RNA input normalization prior to cDNA synthesis and targeted amplification promotes more even distribution of reads across sequencing libraries. For other NEBNext® ARTIC SARS-CoV-2 protocols, please see the NEBNext ARTIC Protocols Collection. To obtain instructions for using NEBNext ARTIC SARS-CoV-2 Primer Mix and the NEBNext® ARTIC SARS-CoV-2 RT-PCR Module please see the NEBNext ARTIC SARS CoV2 RT PCR Module Manual.

Monitoring the cDNA synthesis of dengue-2 virus by RT PCR

1995 ◽  
Vol 51 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Hsiao-Sheng Liu ◽  
Hong-Chang Tzeng ◽  
Cheng-Chen Chen
Keyword(s):  
Rt Pcr ◽  
Cdna Synthesis

scholarly journals Occurrence of Lily mottle virus on Lilium in Italy

Plant Disease ◽  
2012 ◽  
Vol 96 (5) ◽  
pp. 771-771 ◽  
Author(s):  
D. Rizzo ◽  
L. Stefani ◽  
M. Paoli ◽  
S. Lazzereschi ◽  
B. Nesi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leaf Tissue ◽  
Flowering Plant ◽  
Mottle Virus ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Vein Clearing ◽  
Cp Gene ◽  
Necrotic Spots

Lily mottle virus (LMoV), a member of the genus Potyvirus, is one of the main viruses infecting lily. Symptoms on lily differ according to the susceptibility and sensitivity of different cultivars and hybrids. They range from leaf mottle or mosaic, vein clearing, chlorotic and yellow streaking, leaf curling, and necrotic spots, to milder forms of leaf symptoms. Plants may even be symptomless at some stages of growth. A varietal collection of Lilium from the early 1990s is held in Pistoia Province (Tuscany, Italy) and is composed of Asian hybrids obtained from intraspecific breeding of commercial cultivars. During a survey conducted from May to June 2010, several plants showing vein clearing, leaf mottle, leaf mosaic, and reddish brownish necrotic spots were observed. Leaf samples from 60 symptomatic or symptomless lily plants, belonging to 20 cultivars, were collected and tested for the presence of LMoV. Samples were assayed by double-antibody sandwich (DAS)-ELISA and eight of them, belonging to four different cultivars, tested positive. Total RNA was extracted from 2 g of leaf tissue of every collected sample according to the protocol described earlier (3) and cDNA synthesis was performed with an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Samples were tested by reverse transcription (RT)-PCR and real-time PCR assays using primers LMoV1 (5′-GCAAATGAGACACTCAATGCTG-3′) and LMoV2 (5′-CGTGCGTGAAGTAACTTCATAG-3′) designed to amplify 651 bp of the coat protein (CP) gene of LMoV (1). Results obtained with RT-PCR and real-time PCR exactly matched those achieved with ELISA assay, and the eight positive samples showed amplicons of the expected size. PCR products from five infected samples were directly sequenced from both directions and submitted in GenBank (Accessions Nos. JQ655106 to JQ655110). Our isolates share more than 99% nucleotide identity among each other. Comparison with other LMoV-CP gene sequences present in GenBank showed nucleotide identities ranging from 93 to 94% with LMoV isolates from South Korea (GenBank Accession Nos. GQ150683 to GQ150686), China (GenBank Accession Nos. EU348826, AJ748256, AJ564636, and AJ564637), Australia (GenBank Accession No. JN127341), and Japan (GenBank Accession No. AB570195). To our knowledge, this is the first report of LMoV on Lilium in Italy where this virus was already reported to infect escarole (2). Considering the economic importance of Lilium production as a flowering plant in Pistoia Province, and in several other areas of Italy, the report of LMoV present on lilies suggests the use of healthy propagation material and the adoption of preventive measures to avoid its diffusion. References: (1) J.-H. Lim et al. Korean J. Microbiol. 45:251, 2009. (2) V. Lisa et al. Plant Dis. 86:329, 2002. (3) D. J. MacKenzie et al. Plant Dis. 81:222, 1997.

scholarly journals Detection the Ability of Aspergillus flavus Isolated from Wheat Grains for Aflatoxin B1 Production using RT- PCR

2015 ◽  
Vol 9 (2) ◽  
pp. 102-106
Author(s):  
Eman Noaman Ismail
Keyword(s):  
Molecular Weight ◽  
Triticum Aestivum ◽  
Aspergillus Flavus ◽  
Aflatoxin B1 ◽  
Reverse Transcription ◽  
Regulatory Gene ◽  
Rapid Assessment ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Wheat Grains

Aspergillus flavus is aflatoxinogenic and potential aflatoxins producers in agriculturalcommodities. The present study was conducted determine the ability of eleven strains of A. flavusisolated from Iraqi wheat grains Triticum aestivum. The isolates have been detected by molecularmethods using Reverse Transcriptase RTPCR. In this study, RNA was extracted from A. flavus,cDNA synthesis and rapid assessment of eleven isolates of A. flavus was accomplished usingprimer pair for the aflatoxin regulatory gene aflR Reverse transcription-Polymerase chainreaction (RT–PCR).Positive amplification was achieved for all the isolates with a molecularweight 798 to aflR1 and 400bp to aflR2. Also the result of the amplification showed there are nodifferences with the two molecular weight between the 11 isolated strains of A. flavus in theiraflatoxin B1 production, but the first strain differed in their banding florescence as comparedwith others strains this reflect the genetic differences in aflatoxin B1 production between them.

scholarly journals mRNA extraction, cDNA synthesis and tillering specific gene isolation from BLB resistant Binashail rice

2015 ◽  
Vol 1 (2) ◽  
pp. 265-270
Author(s):  
Papia Jahan ◽  
Ahmed Hossain ◽  
KM Nasiruddin ◽  
Sabina Yasmin ◽  
Fahmida Khatun ◽  
...  
Keyword(s):  
Biotic Stress ◽  
Bacterial Blight ◽  
Gene Isolation ◽  
Specific Gene ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Irrigated Rice ◽  
Mrna Extraction ◽  
Bacterial Blight Disease ◽  
Blight Disease

Bacterial Leaf Blight (BLB) caused by Xanthomonas oryzaepv. oryzae (Xoo) bacteria, is a major biotic stress in the irrigated rice belts. Genetic modification is the most effective and economical control for bacterial blight disease. For high yielding, branching capacity is most important. Molecular survey was conducted to identify the presence of tillering specific gene in BLB resistant Binashail rice. Reproducible means of Binashail rice at 21 days was designed based on the timing of full expression of the leaf. PCR with primers specific for tillering specific gene was used in the study. The cDNA of Binashail rice synthesized by an improved RT-PCR technique, derived from the total mRNAs extracted from the plant leaf. Gel documentation showed that the size of the synthesized cDNA was 250bp and tillering specific gene was 201bp in length long. The identification of tillering specific gene in Binashal rice germplasm will help in accelerating the elite breeding program in future.Asian J. Med. Biol. Res. June 2015, 1(2): 265-270

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 Presence of polyadenylated 3’ tail in RNA of Pepper mild mottle virus allows the Oligo(dT)18 in Priming cDNA Synthesis of its genomic RNA template

2020 ◽  
Author(s):  
Nidhi Kumari ◽  
Vivek Sharma ◽  
Sneha Choudhary ◽  
P.N. Sharma
Keyword(s):  
Pcr Amplification ◽  
Bean Common Mosaic Virus ◽  
Mottle Virus ◽  
Infection Cycle ◽  
Genomic Rna ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Pepper Mild Mottle Virus ◽  
Or Gene ◽  
Prime End

AbstractThe PCR amplification of majority of the ssRNA of both genomic and non-genomic mRNA is accomplished by RT-PCR. The mRNA is subjected to cDNA synthesis using reverse transcriptase and either Oligo(dT)18, or random or gene specific reverse primers based priming strategies. The choice of primer largely depends on the nature of 3 prime terminus of mRNA and length of cDNA synthesized. In general, oligo(dT)18 is the preferred choice for mRNAs having poly(A) tail at 3 prime terminus. In general, tobamoviruses lack any poly(A) tail at their 3 prime untranslated region (UTR) which forms a tRNA like structure and upstream pseudoknot domain except tow viruses viz., Hibiscus latent Fort Pierce virus (HLFPV) and Hibiscus latent Singapore virus (HLSV) which accommodate internal poly(A) sequences of 46 and 87 nucleotides long, respectively in their 3 prime UTR. However, determination of full nucleotide sequence of Pepper mild mottle virus (PMMoV) using an oligo(dT)18 primed cDNA as template indicated the libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template which are known to lack poly(A) tail. at the end or internally at its 3 prime end. Moreover, coat protein (CP) gene of PMMoV and bean common mosaic virus (BCMV) (Potyvirus with a poly(A) tract at its 3 prime end) was amplified using cDNA primed with random primer as well as oligo(dT)18 was successfully amplified but with significant variation in the intensity of the amplification band in case of PMMoV but not in BCMV. This clearly indicated the presence of PMMoV mRNA with polyadenylated 3 prime tail in total RNA isolated from PMMoV infected capsicum leaves with abundance of non-polyadenylated PMMoV genomic RNA (gRNA). Hence, we hypothesize that the generation of polyadenylated RNA population during the infection cycle of PMMoV in pepper may be possible reason for libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template isolated from PMMoV infected leaves followed by amplification of entire PMMoV genome through RT-PCR. This is first study indicating the presence of polyadenylated or polyadenylated rich regions in PMMoV gRNA acquired during the infection cycle in pepper.

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