African Swine Fever Virus (ASFV) is a widespread disease which infects members of the pig family (Suidae). A number of tick species are believed to be the vector for the disease, as well as being transmitted by raw pork and pig excrement [1]. After firstly being identified in Kenya in 1921, ASFV became endemic in sub-Saharan Africa, with regular outbreaks being reported across Europe, Asia and South America throughout the century [2]. More recently the virus was introduced in Georgia and spread throughout the region, as well as mass outbreaks occurring in China in 2018 [3].
ASFV is the only member of the Asfaridae family. It is a large enveloped double stranded DNA virus of icosahedral morphology with an average diameter of 200nm and isolates contain genomes between 170-190Kbp encoding for up to 167 open reading frames [2]. The morphology of ASFV consist of several concentric domains. An inner core contains the nucleoid coated with a thick protein layered core shell, which is surrounded by an inner lipid envelope, all of which is encompassed by the capsid [2]. ASFV begins its replication cycle in the nucleus of infected cells before moving to the cytoplasm where the majority of the replication takes place [2]. Genetranscription is highly regulated, with distinct classes of mRNA identified to accumulate at early, intermediate and late transcripts of the virus [2]. The disease induces acute hemorrhagic disease within its hosts, causing high fevers and skin hemorrhages, with death often occurring within ten days of clinical symptoms appearing [4].
The genesig qPCR Detection Kits for ASFV is for the highly sensitive and specific detection of ASFV, including the most recent Asian strain emerging in China (ASFV-SY18), to at least 100 copies of target.
SPECIFICITY:The Primerdesign genesig kit for African Swine Fever Virus (ASFV) is designed for the in vitro quantification of ASFV 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 availableafter the initial design. Primerdesign periodically reviews the detection profiles of our kits and when required releases new versions. The target sequence (p72) has been shown to be a good genetic marker for ASFV in other real time PCR based studies (Tignon et al., 2011). This kit was designed to detect recent isolates from the Chinese outbreak of ASFV and, through the use of multiple probes is predicted to detect strains that have been isolated throughout Europe and Africa.
ASFV specific primer/probe mix (150 reactions; BROWN); FAM labeled
ASFV positive control template (for Standard curve; RED)
Internal extraction control primer/probe mix (150 reactions; BROWN); VIC labeled as standard
Internal extraction control RNA (150 reactions; BLUE)
Endogenous control primer/probe mix (150 reactions; BROWN); FAM labeled
RNAse/DNAse free water (WHITE) for resuspension of primer/probe mixes and internal extraction control RNA
Template preparation buffer (YELLOW) for resuspension of positive control template and standard curve preparation
REAGENTS AND EQUIPMENT TO BE SUPPLIED BY THE USER:
Real-Time PCR Instrument
RNA 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 Lyophilised OneStep or PrecisionTM OneStepPLUS 2x qRT-PCR Mastermix. Contains complete one step qRT-PCR Mastermix
Contains complete one step qRT-PCR MasterMix.
Pipettors and Tips
Vortex and centrifuge
Thin walled 1.5 ml PCR reaction tubes
KIT STORAGE AND STABILITY:This kit is stable at room temperature 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
We do not recommend using the kit after the expiry date stated on the pack.
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 OF TEST:Under optimal PCR conditions genesig ASFV detection kits have very high priming efficiencies of >95% and can detect less than 100 copies of target template.
INSTRUMENTATION REQUIREMENTS:Designed for use with real-time PCR instruments. For use in genesig® q16, look for the “Easy” nomenclature.
ABOUT THE genesig® KITS:genesig® kits are sold for research use only and are not licensed for diagnostic procedures.
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.
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.
Details of the target and priming specificity are included in the individual handbooks above. Packaged, optimized and ready to use.
DNA/RNA EXTRACTION:genesig® Easy DNA/RNA extraction kits are a simple to use, low cost and powerful extraction technology suitable for virtually all sample types.
