The Primerdesign genesig Kit for Bacteriophage MS2 (Bacteriophage MS2) genomes is designed for the in vitro quantification of Bacteriophage MS2 genomes. The kit is designed to have the broadest detection profile possible whilst remaining specific to the Bacteriophage MS2 genome. The primers and probe sequences in this kit have 100% homology with a broad range of Bacteriophage MS2 sequences based on a comprehensive bioinformatics analysis.
Bacteriophage MS2 is a non-enveloped, single-stranded RNA virus that infects “male” Escherichia coli. It is part of the Levivirus genus which is 1 of 2 genera present in the Leviviridae family. The bacteriophages in this family infect their hosts through adsorption to the bacterial pili. MS2’s lack of an envelope renders it resistant to many chemical disinfectants and gives MS2 the ability to endure a range of environmental stresses including changes in temperature. The small MS2 3,569bp genome codes for 4 proteins: assembly, lysis, coat and RNA replicase. The MS2 genome was the first genome to be sequenced fully. The MS2 coat protein (CP) in particular has two known functions: to form the phage coat and to inhibit the translation of the phage replicase gene. It achieves the latter by binding a specific RNA structure. The MS2 phage has 6 main life cycle stages, namely: Adsorption to the cell, Translation of the phage RNA, Replication of the Phage RNA, Synthesis of the A protein, Particle assembly and Cell lysis. Amongst other things, MS2 is both harmless to humans and easy to grow, making it a great model organism for many macromolecular processes. Therefore fast and accurate detection with real time PCR is extremely beneficial.
Bacteriophage MS2 Nucleic acid testing (NAT) is the method of choice for detection and quantification of a wide range of micro organisms. Primerdesign manufactures and supplies high quality quantitative real-time PCR kits for the detection and simultaneous quantification of numerous significant pathogens. A copy number standard curve is provided for quantification and an the internal extraction template (DNA or RNA), controls for the quality of the nucleic acid extraction and eliminates false negative results.
Clinical Relevance:The kit is designed with the broadest possible detection profile to ensure that all clinically relevant strains and subtypes are detected. Target sequences are selected by working with data from key opinion leaders in the field. Multiple sequence alignments and unprecedented real-time PCR expertise in design and validation ensure the best possible kit.
Dynamic Range of Test:Under optimal PCR conditions genesig Bacteriophage MS2 detection kits have very high priming efficiencies of >95% and can detect less than 100 copies of target template.
Test Principle:Real-time PCR A Bacteriophage MS2 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 Bacteriophage MS2 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.
One Step vs. Two Step Real-Time PCR:When detecting/quantifying the presence of a target with an RNA genome Primerdesign recommend the use of a OneStep RT-qPCR protocol. OneStep RT-qPCR combines the reverse transcription and qPCR reaction in a simple closed tube protocol. This saves significant bench time but also reduces errors. The sensitivity of a OneStep protocol is also greater than a two step because the entire biological sample is available to the PCR without dilution. This kit will also work well with a two step approach (Precision nanoScript2 reverse transcription kit and PrecisionPLUS Master Mix) if required but the use of oasig OneStep or PrecisionPLUS OneStep Master Mix is the preferred method.
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 Bacteriophage MS2 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 Bacteriophage MS2 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.
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 notpresent at a high concentration. A separate RT primer mix and a 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 Bacteriophage MS2 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 Bacteriophage MS2 primers. A poor endogenous control signal may indicate that the sample did not contain sufficient biological material.
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.
Kit Components:
Bacteriophage MS2 specific primer/probe mix (150 reactions BROWN)FAM labelled
Bacteriophage MS2 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
Bacteriophage MS2/Internal extraction control/endogenous control RT primer mix (150 reactions GREEN)Required for two step protocol only
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 User:Real-time PCR InstrumentRNA extraction kitThis 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 with minimal PCR inhibitors
oasigTM lyophilized OneStep or Precision®PLUS OneStep 2X RT-qPCR Master MixContains complete OneStep RT-qPCR master mixPipettors and TipsVortex and centrifugeThin walled 1.5 ml PCR reaction tubes
Storage and Stability:This kit is stable at room temperature but should be stored at -20ºC on arrival. Once the lyophilized 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. Primerdesign does not recommend using the kit after the expiry date stated on the pack.
