Method for detecting five types of potato viruses through polyfunctional real-time fluorescent PCR (polymerase chain reaction)
A potato virus and real-time fluorescence technology, which is applied in the fields of fluorescence/phosphorescence, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of affecting the specificity of the experiment, increasing the difficulty of primer design, and the difficulty of distinguishing melting peaks, etc., to achieve Overcoming the effect of low detection sensitivity
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Embodiment 1
[0067] The establishment of embodiment 1, 5 kinds of potato virus basic detection methods
[0068] Step 1: Primer Synthesis
[0069] The primer sequences of 5 kinds of viruses designed in the present invention (see Table 1) were entrusted to Shanghai Sangon Biological Company Technology Service Company to synthesize primers, and the synthesis amount was 2OD per primer.
[0070] Step 2: Small amount of total RNA extraction from positive samples
[0071] Put 500mg of each positive potato dry powder sample containing potato PVX, PVY, PLRV, PVA and PVS virus sources into a 5ml centrifuge tube, then add Trizo at a ratio of 1ml Trizol / 100mg, and place the homogenate on ice for 5min. Add chloroform at a ratio of 200ul chloroform / ml Trizol. Shake vigorously for 30s, place on ice for 5-10min, after layering, centrifuge at 12000r / min for 20min at 4°C, take the supernatant into another 5ml centrifuge tube, add an equal volume of isopropanol (about 0.5ml) to lightly Gently mix, place a...
Embodiment 2
[0081] Potato virus detection in the potato leaves of embodiment 2, Hangzhou Gaosha area
[0082] Step 1: Primer Synthesis
[0083] Same as the first step in Example 1.
[0084] Step 2: Leaf Sampling
[0085] The sampling location is the Gaosha area of Hangzhou, Zhejiang. Select the plants with virus symptoms in the potato planting area, and cut the leaves with scissors. Every time a plant sample is taken, the scissors are disinfected with 75% (volume concentration) alcohol, and then the next sampling is performed. Each sample was packed into a polyethylene food bag, and then put into a sampling box for storage and brought back to the laboratory. A total of 20 samples were set.
[0086] Step 3: small amount of total RNA extraction from leaf samples
[0087] The operation steps are equal to the second step of Example 1.
[0088] Step 4: Reverse transcription to synthesize cDNA
[0089] Same as the third step of Example 1.
[0090] Step 5: Multiplex fluorescent quantit...
Embodiment 3
[0095] Embodiment 3, the detection of potato virus in potato tuber
[0096] Step 1: Primer Synthesis
[0097] Same as the first step in Example 1.
[0098] Step Two: Tuber Sampling
[0099] The sampling location for the detection of potato tuber virus was carried out in Haining, Zhejiang Province. A total of 5 varieties were detected in this experiment. Five plants were randomly selected from each variety, and one tuber was taken from each plant. After the tubers were brought back to the laboratory, the soil was washed away with tap water, then washed three times with double distilled water, and dried at room temperature. Cut 5 tubers of each variety into small pieces and mix them with a knife, then take 500 mg for extraction of total RNA. A total of 20 samples were set.
[0100] Step 3: Mini-extraction of total RNA from potato tuber samples
[0101] Take 500 mg of the above potato tuber sample and grind it into powder in liquid nitrogen, then put it into a 5 ml centrifu...
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