GB/T 22917-2008Protocol of fluorogenic RT-PCR for swine vesicular disease virus (English PDF)

2.1 Fluorescent RT-PCR

Fluorescence reverse transcription - polymerase chain reaction.

The number of cycles when fluorescence signal of each reaction tube reaches a set threshold experienced.

2.3 RNA

RNA.

2.4 DEPC

Dicarbonate phospholipids.

2.5 PBS

Phosphate buffered saline, recipe see Appendix A.

2.6 T Rao enzyme q

T Rao q DNA polymerase.

2.7 S D Vp Vp

Swine vesicular disease virus.

Principle 3

According to a specific gene sequence of swine vesicular disease virus synthesized a pair of specific primers and a dual-labeled probes specific fluorescence. Primers and probe design and through rigorous screening to cover all reported strains of swine vesicular disease virus. 5 'end fluorescent probes labeled FAM Fluorescein, 3 'TAMRA labeled fluorescein, which can absorb at close range within the 5' fluorophore reporter fluorescent signal emitted. Amplification, Since T Rao q enzyme 5 '-> 3' exonuclease activity, when extended to the fluorescent probe, which was cut to two groups separated, quenching effect disappears, fluorescent letter Number generation. Thus, by detecting the fluorescence signal is detected on a nucleic acid template.

4.1 Instruments and Equipment

4.1.1 RT-PCR fluorescence detector.

4.1.2 high-speed desktop refrigerated centrifuge (centrifugal speed 12000r/min or more).

Palm Desktop 4.1.3 or centrifuge (centrifugal speed 3000r/min).

4.1.4 Mixer.

4.1.5 refrigerator (2 °C ~ 8 °C and -20 °C two kinds).

4.1.6 Micro adjustable pipette (5µL, 10µL, 100µL, 1000µL) and supporting filter tips.

4.1.7 1.5mL, 0.5mL siliconized Eppendorf tube. the Eppendorf tubes and emitters containing 0.1% DEPC soaked in distilled water three Overnight, 121 °C ± 2 °C autoclave 15min, 40 °C drying spare. Commercially available Eppendorf tubes and emitters have been suicide, can be directly