PCRmax Hand Instruction Manual
polyprotein gene
Hand, Foot and Mouth
Disease
PCRmax Ltd qPCR test
TM
150 tests
For general laboratory and research use only
Quantification of Hand, Foot and Mouth Disease genomes
Advanced kit handbook HB10.05.09
Published Date: 11/05/2016
1
Hand, foot and mouth disease (HFMD) is a human syndrome caused by intestinal viruses
Human enterovirus C and enterovirus 71 (EV-71). It is moderately contagious and is
spread through direct contact with the mucus, saliva, or feces of an infected person.
Aside from HFMD, EV71 is sometimes associated with severe central nervous system
diseases. It is a non-enveloped, spherical virion, about 30 nm in diameter with a capsid
surrounding the naked RNA genome. The linear genome is single stranded sense RNA of
7.4 kb and is composed of a single ORF encoding a polyprotein.
Human enterovirus C belongs to the genus Enterovirus. The isometric capsid has a
diameter of 28-30 nm and appears round. The genome is not segmented and contains a
single molecule of linear positive-sense, single-stranded RNA consisting of 7400
nucleotides. They are also capable of causing causing herpangina and acute hemorrhagic
conjunctivitis (AHC).
HFMD typically affects children under 10 years of age. Adults and older children with
HFMD tend to develop a milder form of the illness compared with younger children. The
incubation period is 3-5 days. The disease generally presents as a fever, malaise, sore
mouth and development of a rash. Mouth lesions appear on the inside surfaces of the
cheeks, gums and on the sides of the tongue. Raised pink spots that develop into blisters,
which may persist for seven to ten days, can also occur as a rash, especially on the palms,
fingers, soles and occasionally on the buttocks. Attachement of the virus to host receptors
mediates endocytosis of the virus into the host cell. The capsid then undergoes a
conformational change and releases VP4 that opens a pore in the host endosomal
membrane and the viral genomic RNA penetrates into the host cell cytoplasm (the empty
capsid remains intact). VPg is removed from the viral RNA, which is then translated into a
processed polyprotein. Replication occurs in viral factories made of membrane vesicles
derived from the ER. A dsRNA genome is synthesized from the genomic ssRNA(+) and
packaged into preassembled capsids which are then released by cell lysis.
Most cases are passed on by coughing and sneezing which transmits the virus into the air
and outbreaks are reported in nurseries and schools where children are in close proximity
to each other. The virus is also spread by direct contact with nasal and throat secretions or
faeces of the infected person. Infected individuals are often contagious several weeks after
the symptoms have disappeared.
Introduction to Hand, Foot and Mouth Disease
Quantification of Hand, Foot and Mouth Disease genomes
Advanced kit handbook HB10.05.09
Published Date: 11/05/2016
2
Specificity
The PCRmax qPCR Kit for Hand, Foot and Mouth Disease (HFMD) genomes is designed
for the in vitro quantification of HFMD genomes. The kit is designed to have the broadest
detection profile possible whilst remaining specific to the HFMD genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
HFMD sequences based on a comprehensive bioinformatics analysis.
If you require further information, or have a specific question about the detection profile of
this kit then please send an e.mail to help@pcrmax.com and our bioinformatics team will
answer your question.
Quantification of Hand, Foot and Mouth Disease genomes
Advanced kit handbook HB10.05.09
Published Date: 11/05/2016
3
Kit Contents
• Human enterovirus C primer/probe mix (150 reactions BROWN)
FAM labelled
• Human enterovirus 71 primer/probe mix (150 reactions BROWN)
FAM labelled
• Human enterovirus C positive control template (for Standard curve RED)
• Human enterovirus 71 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
• HEV-C and EV-71/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 and internal extraction control RNA
• Template preparation buffer (YELLOW)
for resuspension of positive control templates and standard curve preparation
Reagents and equipment to be supplied by the user
Real-Time PCR Instrument
RNA extraction kit
This kit is designed to work well with all processes that yield high quality RNA with
minimal PCR inhibitors.
Lyophlised 2x RT-qPCR MasterMix
This kit is designed to be compatible with all commercially available OneStep
Mastermixes that run with standard cycling conditions.
Pipettors and Tips
Vortex and centrifuge
Thin walled 1.5 ml PCR reaction tubes
Quantification of Hand, Foot and Mouth Disease genomes
Advanced kit handbook HB10.05.09
Published Date: 11/05/2016
4
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