GB/T 22915-2008Protocol of universal fluorogenic RT-PCR for foot and mouth 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 FMD Vp

FMD virus.

Principle 3

According fragments from various types of FMD virus common gene specific sequence, A pair of universal primers and a common

Specific probe. Fluorescent probes 5 'fluorescein labeled FAM, 3' TAMRA labeled fluorescein, the 3 'end of the quencher in close

Can absorb within the 5 'fluorophore reporter fluorescent signal emitted. However, in amplifying, since the T q Rao enzyme 5 '-> 3' exonuclease activity, extension

When the fluorescent probe, which was cut to two groups separated, quenching effect disappears, the fluorescence signal. Therefore, by detecting the fluorescence signal

Template nucleic acid is detected.

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).

4.1.3

tabletop 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

Eppendorf tubes (1.5mL), a transparent thin-walled PCR tube (0.2mL).