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SN/T 0762-2011: Protocol of real-time RT-PCR for detection of classical swine fever virus
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SN/T 0762-2011: Protocol of real-time RT-PCR for detection of classical swine fever virus

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Protocol of real-time RT-PCR for detection of classical swine fever virus People's Republic of China Entry-Exit Inspection and Quarantine Standards Classical swine fever virus fluorescent RT-PCR detection method Issued on. 2011-05-20 2011-12-01 implementation People's Republic of China The State Administration of Quality Supervision, Inspection and Quarantine released

Foreword

This standard was drafted in accordance with GB/T 1.1-2009 given rules. This standard is proposed and managed by the National Certification and Accreditation Administration Committee. This standard was drafted. People's Republic of China Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai Fosun Medical Technology Development Co., Ltd. The main drafters of this standard. Li Chunyang, Liu Junping, Xiong Wei, a single pine Hua, Huang Zhongrong, Xia Yi, Hu Yongqiang, Li Shuqing, Wang Qiao whole. Classical swine fever virus fluorescent RT-PCR detection method

1 Scope

This standard specifies the method of operation of classical swine fever virus fluorescent RT-PCR detection. This standard applies to pigs and swine fever virus detection products.

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

3 Abbreviations

The following abbreviations apply to this document. Fluorescent RT-PCR. fluorescent RT - polymerase chain reaction The number of cycles the amount of fluorescence signal of each reaction tube reaches a set threshold when experienced. Ct values RNA. ribonucleic acid DEPC. Coke ethylene carbonate Taq enzyme. TaqDNA polymerase Principle 4 Classical swine fever virus is enveloped positive-strand RNA viruses. According to the conserved sequences of different isolates of classical swine fever virus gene, a pair of specific Primers and a dual-labeled probes specific fluorescence. Probe and conserved sequences of viral genes combine, the binding site is located primers Area. Probe 5 'end labeled FAM luciferin, it emits can be received detection equipment, the report called fluorophores (with R represents), 3 'TAMRA labeled fluorescein, which can absorb at close range within the 5' fluorophore reporter fluorescent signal emitted, called quenching Off fluorophore (denoted by Q). When the PCR reaction into the annealing stage, primers and probes simultaneously with the target gene binding, then probe R groups on the fluorescence signal emitted is absorbed by the Q group, the instrument can not detect the fluorescence signal; PCR reaction is carried out to the extended stage, Taq Enzymes play its 5 '→ 3' exonuclease enzyme hydrolysis probe function, at the same time marked on the probe freed R groups, the R Q fluorescence emitted no longer be absorbed and is received by the detector. With the growth of performing PCR reactions, the PCR product with fluorescence signal Was correspondence. 5 equipment, materials and reagents 5.1 equipment, materials 5.1.1 PCR fluorescence detector. 5.1.2 high-speed desktop refrigerated centrifuge (centrifugal velocity 12000g above). 5.1.3 ordinary desktop centrifuge (centrifugal speed 3000g). ...