GB/T 27538-2011Animal influenza detection - Method of pyrosequencing for HA and NA in influenza Virus A (H1N1) (English PDF)

1 Scope

This standard specifies the pyrosequencing technique for Type A H1N1 influenza virus mutants in Mexico and the HA and NA nucleic acid

Sequence Detection.

This standard applies to nucleic acid detection type A H1N1 influenza virus type A H1N1 influenza and common virus variants Mexico.

2 Normative references

The following documents for the application of this document is essential. For dated references, only the dated version suitable for use herein

Member. For undated references, the latest edition (including any amendments) applies to this document.

Laboratory use specifications and test methods GB/T 6682 Analysis

GB/T 18088 Entry and Exit Animal Quarantine sampling

GB 19489 General requirements for laboratory biosafety

NY/T 541 animal disease laboratory sampling method

SN/T 1193 genetic testing laboratory technical requirements

3 Abbreviations

The following abbreviations apply to this document.

ATP. adenosine triphosphate (adenosinetriphosphate)

DNA. deoxyribonucleic acid (deoxyribonucleicacid)

DEPC. diethyl pyrocarbonate (diethylpyrocarbonate)

PBS. phosphate buffered saline (phosphatebelancedsolution)

Pyrosequencing. Pyrosequencing

RT-PCR. reverse transcription-polymerase chain reaction (reversetranscriptionpolymerasechainreaction)

RNA. ribonucleic acid (ribonucleicacid)

Taq enzyme. TaqDNA polymerase

Principle 4

Pyrosequencing is a single-stranded DNA template was amplified by PCR and sequencing primer hybridization, and DNA polymerase, ATP sulfuric acid enzymes, fluorescent

Luciferase, adenosine triphosphate and dual phosphatase substrate (APS), fluorescein incubation, each added four kinds of dNTPs (dATP, dTTP, dCTP, of dGTP), and as the template pair, this end of the primer dNTP form a covalent bond, a group releasing pyrophosphate (PPi); ATP sulfurylase in

The case of the presence of a catalytic APS pyrophosphate formed ATP, ATP driven luciferase-mediated oxidation of luciferin-converting phosphor to oxide phosphor

Optical element emits visible light signal proportional to the amount of ATP; the optical signal from the CCD detector (CCD) and collected by the software into peaks.

Nucleotide number is proportional to the peak height of the response signal in each optical incorporated. ATP and unincorporated dNTP by the adenosine triphosphate diphosphate

Enzymatic degradation, quenched optical signal regeneration and reaction system.

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