Development of a New Internally Controlled One-Step Real-Time RT-PCR for the Molecular Detection of Enterovirus A71 in Africa and Madagascar
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Development of a New Internally Controlled One-Step Real-Time RT-PCR for the Molecular Detection of Enterovirus A71 in Africa and Madagascar

- August 24, 2021 | August 26, 2021 - Djoumana

Enterovirus A71 (EV-A71) is a number one reason behind hand-foot-and-mouth illness (HFMD) and might be related to extreme neurological problems. EV-A71 strains might be labeled into seven genogroups, A-H, on the idea of the VP1 capsid protein gene sequence. Genogroup A contains the prototype pressure; genogroups B and C are accountable of main outbreaks worldwide, however little is understood in regards to the others, notably genogroups E and F, which have been not too long ago recognized in Africa and Madagascar, respectively. The circulation of EV-A71 within the African area is poorly recognized and possibly underestimated. A speedy and particular assay for detecting all genogroups of EV-A71 is required.

On this examine, we developed a real-time RT-PCR assay with a aggressive inner management (IC). The primers and TaqMan probe particularly goal the genomic area encoding the VP1 capsid protein. Various EV-A71 RNAs have been efficiently amplified from the genogroups A, B, C, D, E, and F, with related sensitivity and strong reproducibility. Neither cross response with different EVs nor main interference with the aggressive IC was detected. Experimentally spiked stool and plasma specimens offered constant and reproducible outcomes, and validated the usefulness of the IC for demonstrating the presence of PCR inhibitors in samples.

The evaluation in an African laboratories community of 1889 untyped enterovirus isolates detected 15 EV-A71 of various genogroups. This particular real-time RT-PCR assay supplies a strong and delicate methodology for the detection of EV-A71 in organic specimens and for the epidemiological monitoring of EV-A71 together with its not too long ago found genogroups.

Formalin-fixed paraffin-embedded (FFPE) tissues are an vital supply for investigation of dengue virus (DENV) an infection, notably when blood or recent frozen (FF) samples are unavailable. Histopathologic options and immunohistochemistry could have poor sensitivity and serotype willpower is just not at all times doable. Viral RNA genome detection assessments are sooner and regarded probably the most delicate approach for this sort of evaluation, nonetheless, the usage of molecular strategies utilized to FFPE tissues remains to be restricted.

The authors utilized a single-step multiplex reverse transcription-quantitative polymerase chain response (RT-qPCR) for the investigation of DENV an infection and typing to FFPE samples of 32 deadly circumstances acquired throughout the 2019 outbreak that occurred in São Paulo state, Brazil. The authors in contrast the outcomes with these obtained utilizing FF tissues. Of the 24 circumstances with each FF and FFPE samples, 22 (91.67%) of the FF and 19 (76.20%) of the FFPE specimens have been optimistic. Two circumstances (8.33%) examined unfavourable in each forms of samples.

A one–step, one-tube real-time RT–PCR primarily based assay with an automatic evaluation for detection of SARS-CoV-2

Early analysis of SARS-CoV-2 contaminated sufferers is important to regulate the dynamics of the COVID-19 pandemic. We develop a speedy and correct one-step multiplex TaqMan probe-based real-time RT-PCR assay, together with a computational software to systematically analyse the info. Our assay may detect to a restrict of 15 copies of SARS-CoV-2 transcripts-based on experiments carried out by spiking whole human RNA with in vitro synthesized viral transcripts. The assay was evaluated by performing 184 validations for the SARS-CoV-2 Nucleocapsid gene and human RNase P as an inner management reference gene with dilutions starting from 1-100 ng for human RNA on a cohort of 26 medical samples. 5 of 26 sufferers have been confirmed to be contaminated with SARS-CoV-2, whereas 21 examined unfavourable, in keeping with the requirements.

The accuracy of the assay was discovered to be 100% delicate and 100% particular primarily based on the 26 medical samples that must be additional verified utilizing a lot of medical samples. In abstract, we current a speedy, simple to implement real-time PCR primarily based assay with automated evaluation utilizing a novel COVID qPCR Analyzer software with graphical person interface (GUI) to research the uncooked qRT-PCR information in an unbiased method at a value of underneath $Three per response and turnaround time of lower than 2h, to allow in-house SARS-CoV-2 testing throughout laboratories.

Development of a New Internally Controlled One-Step Real-Time RT-PCR for the Molecular Detection of Enterovirus A71 in Africa and Madagascar

The nucleotide area within the ORF1ab gene that has the best variations between the human coronavirus and the bat coronavirus was chosen. Primers have been designed after that sequence. All diagnostic primers are species-specific because the 3´ finish of the sequence differs from that of different species. A primer set additionally creates an artificial optimistic management. Amplified merchandise have been seen in a 2.5% agarose gel, in addition to in an SYBR Inexperienced-Based mostly Actual-Time RT-PCR.

A easy methodology for detection of a novel coronavirus (SARS-CoV-2) utilizing one–step RT–PCR adopted by restriction fragment size polymorphism

A novel coronavirus related to acute respiratory illness (named SARS-CoV-2) is not too long ago recognized in Wuhan metropolis, China, unfold quickly worldwide. Early identification of this novel coronavirus by molecular instruments is essential for surveillance and management of the epidemic outbreak. We aimed to determine a easy methodology for the detection of SARS-CoV-2 in differentiating with SARS-CoV. Primers of our in-house reverse transcription polymerase chain response (RT-PCR) assays have been designed to focus on conserved areas of the RdRP gene and E gene, chosen restriction enzymes EcoRI, Tsp45I, and AluI to differentiate between SARS-CoV-2 and SARS-CoV.

Probe One-Step qRT-PCR SuperMix
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Green One-Step qRT-PCR SuperMix
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Probe One-Step qRT-PCR SuperMix (GC Rich)
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Green One-Step qRT-PCR SuperMix (GC Rich)
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Green Two-Step qRT-PCR SuperMix
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Green miRNA Two-Step qRT-PCR SuperMix
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Green Two-Step qRT-PCR SuperMix (GC Rich)
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PCR SuperMix
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One-Step RT-PCR SuperMix (+dye) (4 kb)
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One-Step RT-PCR SuperMix (+dye) (8 kb)
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One-Step RT-PCR SuperMix (+dye) (8 kb)
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PCR SuperMix (+dye)
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PCR SuperMix (-dye)
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Plus PCR SuperMix
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Pro QPCR SuperMix Kit
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Pro QPCR SuperMix Kit
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T PCR SuperMix (-dye)
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T PCR SuperMix (+dye)
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EasyPfu PCR SuperMix (-dye)
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FastPfu PCR SuperMix (-dye)
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Two-Step RT-PCR SuperMix (12 kb)
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Two-Step RT-PCR SuperMix (15 kb)
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PCR SuperMix for PAGE (+dye)
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Pro QPCR SuperMix Kit - ROX premixed
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Pro QPCR SuperMix Kit - ROX premixed
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Fast Pro QPCR SuperMix Kit - ROX premixed
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Fast Pro QPCR SuperMix Kit - ROX premixed
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One-Step gDNA Removal and cDNA Synthesis SuperMix
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High Fidelity (HiFi) PCR SuperMix (-dye) (DNA, PCR)
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One-Step gDNA Removal and cDNA Synthesis SuperMix (15kb)
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One Step MultiPlex qRT PCR Kit with Probe
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One-Step gDNA Removal and cDNA Synthesis SuperMix (8 kb)
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One-Step gDNA Removal and cDNA Synthesis SuperMix (12 kb)
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Eva QPCR SuperMix Kit
K5052200 200 reactions
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Eva QPCR SuperMix Kit
K5052400 400 reactions
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HiScript II One Step qRT-PCR Probe Kit
Q222-01 100 rxn (50 μl/rxn)
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HiScript II One Step qRT-PCR Probe Kit
Q223-EN01 250 rxns (20 μl/rxn)
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First-Strand cDNA Synthesis SuperMix for PCR
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HiScript III One Step qRT-PCR Probe Kit
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HiScript III One Step qRT-PCR Probe Kit
Q225-EN02 1,000 rxns
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HiScript III One Step qRT-PCR Probe Kit
Q225-EN03 5,000 rxns
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Fast Eva QPCR SuperMix Kit
K5052002 200 rxn
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Power Eva QPCR SuperMix Kit
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Power Eva QPCR SuperMix Kit
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All-in-One First-Strand cDNA Synthesis SuperMix for PCR
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HiScript II U+ One Step qRT-PCR Probe Kit
Q223-01 100 rxn (50 μl/rxn)
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OneScriptcDNA Synthesis SuperMix
G451 25 x 20 ul reactions
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OneScriptcDNA Synthesis SuperMix
G452 100 x 20 ul reactions
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HiScript II One Step qRT-PCR SYBR Green Kit
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Probe qPCR SuperMix
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Evo? cDNA Supermix
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Novo? cDNA Supermix
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Green qPCR SuperMix
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OneScriptPlus cDNA Synthesis SuperMix
G453 25 x 20 ul reactions
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OneScriptPlus cDNA Synthesis SuperMix
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High Fidelity (HiFi) PCR SuperMix (-dye) (lambda DNA, cDNA and plasmid DNA)
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cDNA Synthesis SuperMix
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Green qPCR SuperMix UDG
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Top Green qPCR SuperMix
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Tip Green qPCR SuperMix
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All-in-One cDNA Synthesis SuperMix
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All-in-One cDNA Synthesis SuperMix
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Description: All-in-One cDNA Synthesis SuperMix contains all the necessary components pre-blended in one tube for reverse transcription.

Library Amplification SuperMix
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HiScript III One Step qRT-PCR Probe 5 × Master Mix
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HiScript III One Step qRT-PCR Probe 5 × Master Mix
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HiScript III One Step qRT-PCR Probe 5 × Master Mix
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cDNA Synthesis SuperMix for qPCR
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Top Green qPCR SuperMix (+Dye II)
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Tip Green qPCR SuperMix (+Dye II)
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Library Quantification qPCR SuperMix
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HiScript II Q RT SuperMix for qPCR
R222-01 100 rxn (20 μl/rxn)
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Green Dye One Step qRT-PCR Master Mix For Quantitative Real Time PCR With ROX Dye
MB1004-100Reactions 100 Reactions
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Fly First-Strand cDNA Synthesis SuperMix
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cDNA Synthesis SuperMix for qPCR (GC-rich)
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miRNA First-Strand cDNA Synthesis SuperMix
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HiScript III All-in-one RT SuperMix Perfect for qPCR
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HiScript II Q Select RT SuperMix for qPCR
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HiScript III RT SuperMix for qPCR (+g DNA wiper)
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One-Step TaqProbe qRT-PCR-ROX
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One-Step BrightGreen qRT-PCR-ROX
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One-Step TaqProbe qRT-PCR-iCycler
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One-Step BrightGreen qRT-PCR-iCycler
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First-Strand cDNA Synthesis SuperMix (cDNA up to 12 kb)
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First-Strand cDNA Synthesis SuperMix (cDNA up to 15 kb)
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HiScript II Q Select RT SuperMix for qPCR (+g DNA wiper)
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One Step RT PCR Kit
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One-Step TaqProbe qRT-PCR-No Dye
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One-Step TaqProbe qRT-PCR-Low ROX
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One-Step BrightGreen qRT-PCR-No Dye
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One-Step BrightGreen qRT-PCR-Low ROX
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ExCellenCT One-Step TaqProbe qRT-PCR-ROX
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Green-2-Go 1-Step qRT-PCR Mastermix-iCycler
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ExCellenCT One-Step BrightGreen qRT-PCR-ROX
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ExCellenCT One-Step TaqProbe qRT-PCR-iCycler
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ExCellenCT One-Step BrightGreen qRT-PCR-iCycler
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T-Pro Gel/PCR DNA Purification Mini Kit (100)
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T-Pro Gel/PCR DNA Purification Mini Kit (250)
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T-Pro Gel/PCR DNA Purification Maxi Kit (10)
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Green-2-Go 1-Step qRT-PCR Mastermix-Low Rox
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ExCellenCT One-Step TaqProbe qRT-PCR-No Dye
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ExCellenCT One-Step TaqProbe qRT-PCR-Low ROX
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EUR 212.4

ExCellenCT One-Step BrightGreen qRT-PCR-No Dye
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EUR 212.4

ExCellenCT One-Step BrightGreen qRT-PCR-Low ROX
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EUR 212.4

Green-2-Go 1-Step qRT-PCR Mastermix-Rox (100X20ul Rxn)
QPCR010-R 100Rxn
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One Step RT PCR Kit (cDNA Synthesis + PCR In One Reaction)
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Green-2-Go 1-Step qRT-PCR Mastermix-No Dye (100X20ul Rxn)
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One Step AMV RT-PCR Kit
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AccurSTART One Step RT-qPCR Super PreMix (ONE TUBE)
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AccurSTART One Step RT-qPCR Super PreMix (ONE TUBE)
Q621-EN02 1,000 rxns (20 μl/rxn)
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AccurSTART One Step RT-qPCR Super PreMix (ONE TUBE)
Q621-EN03 10,000 rxns (20 μl/rxn)
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One Step M-MULV RT-PCR Kit
BS665 100preps
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HiScipt II One Step RT-PCR Kit
P611-01 50 rxn (50 μl/rxn)
EUR 234

Green-2-Go 1-Step TaqProbe qRT-PCR Mastermix-no Dye (100X20ul Rxn)
QPCR008-NODYE 100Rxn
EUR 341.88

igScript One Step RT-PCR Kit - 100 Reactions
4211 1/EA
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igScript One Step RT-PCR Kit - 500 Reactions
4213 1/EA
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T-Pro BCA Protein Assay kit
JB04-D001 500assay/KIT
EUR 244.8

HiScipt II One Step RT-PCR Kit (Dye Plus)
P612-01 50 rxn (50 μl/rxn)
EUR 234

T-Pro Bradford Protein Assay kit(1X)
JB04-D002 500ml/BT
EUR 193.2

T-Pro Plasmid Mini Kit (100)
RB94-YPD100 100preps/Kit
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T-Pro Plasmid Mini Kit (250)
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T-Pro Plasmid Midi Kit (20)
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T-Pro Plasmid Maxi Kit (10)
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EUR 266.4

T-Pro Genomic DNA Mini Kit (100)
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EUR 255.6

T-Pro Genomic DNA Midi Kit (20)
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EUR 266.4

T-Pro LumiFast Plus Chemiluminescence Detection Kit (ECL Kit)
JT96-K002M 250ml*2/Kit
EUR 244.8

T-Pro LumiFast Plus Chemiluminescence Detection Kit (ECL Kit)
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T-Pro LumiLong Plus Chemiluminescence Detection Kit (ECL Kit)
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EUR 286.8

T-Pro LumiLong Plus Chemiluminescence Detection Kit (ECL Kit)
JT96-K004S 100ml*2/Kit
EUR 204

T-Pro Plant Genomic DNA Mini Kit (100)
RB94-PGS100 100preps/kit
EUR 266.4

T-Pro Plant Genomic DNA Midi Kit (20)
RB94-PGM020 20preps/kit
EUR 266.4

T-Pro Endotoxin Removal Plasmid Midi kit (25)
RB94-EPI020 20preps/Kit
EUR 297.6

T-Pro Endotoxin Removal Plasmid Maxi kit (10)
RB94-EPM010 10preps/Kit
EUR 318

ranavirus One-Step PCR kit
Oneq-V337-100D 100T
EUR 1039.2

ranavirus One-Step PCR kit
Oneq-V337-150D 150T
EUR 1177.2

ranavirus One-Step PCR kit
Oneq-V337-50D 50T
EUR 861.6

Sapovirus One-Step PCR kit
Oneq-H630-100R 100T
EUR 1932

Sapovirus One-Step PCR kit
Oneq-H630-150R 150T
EUR 2646

Sapovirus One-Step PCR kit
Oneq-H630-50R 50T
EUR 1410

Entamoeba One-Step PCR kit
Oneq-H683-100D 100T
EUR 1039.2

Entamoeba One-Step PCR kit
Oneq-H683-150D 150T
EUR 1177.2

Entamoeba One-Step PCR kit
Oneq-H683-50D 50T
EUR 861.6

Echovirus One-Step PCR kit
Oneq-H722-100D 100T
EUR 1039.2

Echovirus One-Step PCR kit
Oneq-H722-150D 150T
EUR 1177.2

Echovirus One-Step PCR kit
Oneq-H722-50D 50T
EUR 861.6

Mengovirus One-Step PCR kit
Oneq-H453-100R 100T
EUR 1932

Mengovirus One-Step PCR kit
Oneq-H453-150R 150T
EUR 2646

Mengovirus One-Step PCR kit
Oneq-H453-50R 50T
EUR 1410

Astrovirus One-Step PCR kit
Oneq-H491-100R 100T
EUR 1932

Astrovirus One-Step PCR kit
Oneq-H491-150R 150T
EUR 2646

Astrovirus One-Step PCR kit
Oneq-H491-50R 50T
EUR 1410

Adenovirus One-Step PCR kit
Oneq-H501-100D 100T
EUR 1039.2

Adenovirus One-Step PCR kit
Oneq-H501-150D 150T
EUR 1177.2

Adenovirus One-Step PCR kit
Oneq-H501-50D 50T
EUR 861.6

On this report, a 396-bp fragment of the RdRp gene and 345-bp fragment of the E gene have been amplified by one-step RT-PCR. Enzyme Tsp45I cuts the RdRP-amplified product of SARS-CoV-2 producing three fragments of 45, 154, and 197 bp, but it surely didn’t reduce the amplicon of SARS-CoV. In distinction, the amplified product of SARS-CoV was digested with EcoRI producing two fragments of 76 and 320 bp, whereas the amplicon of SARS-CoV-2 was undigested by Tsp45I assist to differentiate clearly SARS-CoV-2 from SARS-CoV on gel electrophoresis.

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