2019-nCoV RNA dependant RNA polymerase (RdRp), genesig Advanced Kit|150 tests||Feature Summary:|1. Rapid detection of 2019-nCoV|2. Positive copy number standard curve for quantification|3. Highly specific detection profile|4. High priming efficiency|5. Broad dynamic detection range (>6 logs)|6. Sensitive to < 100 copies of target|7. Accurate controls to confirm findings||Introduction to 2019-nCoV:|On 31 December 2019, the World Health Organisation (WHO) was alerted of an outbreak of pneumonia in Wuhan City, China, caused by an unknown virus. The virus was sequenced and identified as a novel strain of Coronavirus one week later on 7 January 2020. It belongs to the same family of viruses which include Severe Acute Respiratory Syndrome (SARS) and Middle Eastern Respiratory Syndrome (MERS).|2019-nCoV is a positive-sense, single-stranded RNA virus which is thought to be of zoonotic origin. Symptoms of those infected include fever, dry cough, fatigue, shortness of breath and respiratory distress. Additionally, cases have resulted in renal failure, pneumonia and death. Those most seriously affected are individuals with already impaired immune systems, however approximately one quarter of otherwise well patients have required intensive care upon hospital admission.|Human-to-human transmission of the virus has been confirmed, with previous Coronavirus outbreaks (such as SARS) originating from similar environments in Asian markets, where humans and live animals are in close proximity.||Specificity:|The Primerdesign genesig Kit for 2019-nCoV (2019-nCoV) genomes is designed for the in vitro quantification of 2019-nCoV genomes. The kit is designed to have a broad detection profile. Specifically, the primers represent 100% homology with over 95% of the NCBI database reference sequences available at the time of design.|The dynamics of genetic variation means that new sequence information may become available after the initial design. Primerdesign periodically reviews the detection profiles of our kits and when required releases new versions.|The Primerdesign genesig Kit for 2019-nCoV detection is designed for the in vitro quantification of 2019-nCoV genomes. The primers and probe are designed to have 100% homology with the 16 genome sequences available on the GISAID database as of 23 January 2020, some of which were subsequently available on NCBI. The primers and probe target the RdRp gene which has previously been used in the identification of the SARS coronavirus, however there is no cross reactivity with this or any other coronavirus sequenced thus far.||Kit Contents:|• 2019-nCoV specific primer/probe mix (150 reactions BROWN) FAM labelled|• 2019-nCoV positive control template (for Standard curve RED)|• Internal extraction control primer/probe mix (150 reactions BROWN)|VIC labelled as standard|• Internal extraction control RNA (150 reactions BLUE)|• Endogenous control primer/probe mix (150 reactions BROWN) FAM labelled|• RNase/DNase free water (WHITE)|for resuspension of primer/probe mixes|• Template preparation buffer (YELLOW)|for resuspension of internal control template, positive control template and standard curve preparation||Reagents and equipment to be supplied by the user:|Real-time PCR Instrument||Extraction kit (supplied separately):|OasigTM lyophilised OneStep or Precision®PLUS OneStep 2X RT-qPCR Master Mix:|Contains complete OneStep RT-qPCR master mix|Note:|This kit is recommended for use with genesig Easy DNA/RNA Extraction kit. However, it is designed to work well with all processes that yield high quality RNA and DNA with minimal PCR inhibitors.||Pipettors and Tips|Vortex and centrifuge|Thin walled 1.5 ml PCR reaction tubes||Kit Storage and Stability:|This kit is stable at RT but should be stored at -20°C on arrival. Once the lyophilised components have been resuspended they should not be exposed to temperatures above -20°C for longer than 30 minutes at a time and unnecessary repeated freeze/thawing should be avoided.||The kit is stable for six months from the date of resuspension under these circumstances.||If a standard curve dilution series is prepared this can be stored frozen for an extended period. If you see any degradation in this serial dilution a fresh standard curve can be prepared from the positive control.||Suitable Sample Material:|All kinds of sample material suited for PCR amplification can be used. Please ensure the samples are suitable in terms of purity, concentration, and RNA/DNA integrity (An internal PCR control is supplied to test for non specific PCR inhibitors). Always run at least one negative control with the samples. To prepare a negative-control, replace the template RNA sample with RNase/DNase free water.||Dynamic Range:|Under optimal PCR conditions genesig 2019-nCoV detection kits have very high priming efficiencies of >90% and can detect less than 100 copies of target template.||Trademarks:|PrimerdesignTM is a trademark of Primerdesign Ltd.|genesig® is a registered trademark of Primerdesign Ltd.|The PCR process is covered by US Patents 4,683,195, and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche AG. BI, ABI PRISM® GeneAmp® and MicroAmp® are registered trademarks of the Applera Genomics (Applied Biosystems Corporation). BIOMEK® is a registered trademark of Beckman Instruments, Inc.; iCyclerTM is a registered trademark of Bio-Rad Laboratories, Rotor- Gene is a trademark of Corbett Research. LightCyclerTM is a registered trademark of the Idaho Technology Inc. GeneAmp®, TaqMan® and AmpliTaqGold® are registered trademarks of Roche Molecular Systems, Inc., The purchase of the Primerdesign TM reagents cannot be construed as an authorization or implicit license to practice PCR under any patents held by Hoffmann-LaRoche Inc.||Notices and Disclaimers:|This product is developed, designed and sold for research purposes only. It is not intended for human diagnostic or drug purposes or to be administered to humans unless clearly expressed for that purpose by the Food and Drug Administration in the USA or the appropriate regulatory authorities in the country of use.||Principles of the Test:|Real-time PCR:|A 2019-nCoV specific primer and probe mix is provided and this can be detected through the FAM channel.|The primer and probe mix provided exploits the so-called TaqMan® principle. During PCR amplification, forward and reverse primers hybridize to the 2019-nCoV cDNA. A fluorogenic probe is included in the same reaction mixture which consists of a DNA probe labeled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved and the reporter dye and quencher are separated. The resulting increase in fluorescence can be detected on a range of qPCR platforms.||Positive Control:|For copy number determination and as a positive control for the PCR set up, the kit contains a positive control template.|This can be used to generate a standard curve of 2019-nCoV copy number / Cq value. Alternatively the positive control can be used at a single dilution where full quantitative analysis of the samples is not required. Each time the kit is used, at least one positive control reaction must be included in the run. A positive result indicates that the primers and probes for detecting the target 2019-nCoV gene worked properly in that particular experimental scenario. If a negative result is obtained the test results are invalid and must be repeated. Care should be taken to ensure that the positive control does not contaminate any other kit component which would lead to false-positive results. This can be achieved by handling this component in a Post PCR environment. Care should also be taken to avoid cross-contamination of other samples when adding the positive control to the run. This can be avoided by sealing all other samples and negative controls before pipetting the positive control into the positive control well.||Negative Control:|To validate any positive findings a negative control reaction should be included every time the kit is used. For this reaction the RNase/DNase free water should be used instead of template. A negative result indicates that the reagents have not become contaminated while setting up the run.||Internal RNA Extraction Control:|When performing RNA extraction, it is often advantageous to have an exogenous source of RNA template that is spiked into the lysis buffer. This control RNA is then co-purified with the sample RNA and can be detected as a positive control for the extraction process. Successful co-purification and qPCR for the control RNA also indicates that PCR inhibitors are not present at a high concentration.|A separate qPCR primer/probe mix are supplied with this kit to detect the exogenous RNA using qPCR. The PCR primers are present at PCR limiting concentrations which allows multiplexing with the target sequence primers. Amplification of the control cDNA does not interfere with detection of the 2019-nCoV target cDNA even when present at low copy number. The Internal control is detected through the VIC channel and gives a Cq value of 28+/-3 depending on the level of sample dilution.||Endogenous Control:|To confirm extraction of a valid biological template, a primer and probe mix is included to detect an endogenous gene. Detection of the endogenous control is through the FAM channel and it is NOT therefore possible to perform a multiplex with the 2019-nCoV primers. A poor endogenous control signal may indicate that the sample did not contain sufficient biological materialImportant Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. Toxicity and Hazards: All products should be handled by qualified personnel only, trained in laboratory procedures.

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