GB/T 36820-2018Detection method of Sugarcane streak mosaic virus by real-time reverse transcription polymerase chain reaction (RT-PCR) (English PDF)

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009.

1 Scope

This standard specifies the sugarcane stripe mosaic virus (Sugarcanestreakmosaicvirus) real-time fluorescent reverse transcription polymerase chain reaction Instruments and equipment, reagents and materials, sample collection and pre-processing, detection, and judgment and presentation of results should be performed (RT-PCR).

This standard applies to the rapid detection and diagnosis of living host plants that may have sugarcane stripe mosaic virus.

2 Abbreviations

The following abbreviations apply to this document.

CP. coat protein

Ct value. the number of cycles experienced by the fluorescent signal in each reaction tube reaching the set threshold in a real-time fluorescent PCR reaction (cycle Threshold)

cDNA. single-stranded DNA (complementaryDNA) complementary to the RNA strand

ExTaqHS enzyme. hotstart DNA polymerase (hotstartDNApolymerase)

FAM. 6-carboxy-fluorescein

PCR. polymerase chain reaction (polymerase chain reaction)

RNA. ribonucleic acid

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

SCSMV. Sugarcane Stripe Mosaic Virus (Sugarcanestreakmosaicvirus)

TAMRA. 6-carboxytetramethylrhodamine

Tip. Tip

3 Principle of the method

Design a pair of specific primers and a special one that are conserved only between the CP genes of the virus according to the CP gene sequence of sugarcane stripe mosaic virus A heterologous fluorescent double-labeled hydrolyzed oligonucleotide probe (TaqMan probe). First reverse the viral RNA with reverse transcriptase cDNA, and then real-time fluorescent PCR amplification reaction in the same reaction tube using cDNA as a template, hydrolyzed oligonucleotide probe The fluorescence signal intensity of the (TaqMan probe) is proportional to the increase of the PCR product of the target sequence, and is amplified by collecting PCR.

By increasing the fluorescence signal value of the process, it is possible to determine whether the sample carries the target sequence. See Appendix A for information on sugarcane streak mosaic disease.

4 Instruments and equipment

4.1 Real-time fluorescent PCR detector. excitation/detection wavelength range 350nm~750nm; available fluorescent dye (SYBRGreenI) and water Decoded oligonucleotide probe (TaqMan probe); temperature rise and fall >=2.0 °C/s; uniformity ±0.5 °C; accuracy ±0.3 °C; temperature The range is from 4 °C to 100 °C.

4.2 Nucleic acid protein analyzer or UV spectrophotometer.

4.3 Electronic balance. Sensitivity 0.01g.