A pair of Paraleyrodes pseudonaranjae Martin specific SS-COI primers, rapid PCR detection method and kit
A detection kit and detection method technology, applied in the field of molecular biology, can solve the problems of reducing product yield and quality, hazards, deformities, etc., and achieve the effects of improving accuracy and sensitivity, high detection accuracy, and saving detection time
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Embodiment 1
[0028] Example 1: Amplification effect of primers PPZYF / PPZYR on whitefly
[0029] 1) Preparation of whitefly template DNA
[0030]Place the single-headed whitefly on a parafilm dripped with 20 μL extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0), and use the bottom of a 0.2 mL PCR tube as a homogenizer to thoroughly grind , transfer the homogenate into a 1.5mL centrifuge tube with a micropipette; then wash the homogenizer twice with 200μL buffer solution, transfer to the same centrifuge tube, mix well, add 5μL proteinase K (20mg / mL), mix thoroughly Water bath at 60°C for 1 hour (mix once in the middle); then bath in boiling water for 8 minutes, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, mix gently for dozens of times, and place on ice for 30 minutes ; Centrifuge at 4°C, 12000r / min for 20min, take the supernatant, add 440μL pre-cooled absolute ethanol, mix gently, and wait for a small amount of flocculent precipitate to stand at -20°C...
Embodiment 2
[0047] Example 2: Amplification effect of primers PPZYF / PPZYR on different stages and sexes of whitefly A.
[0048] 1) Preparation of the template DNA of the double-hooked whitefly
[0049] Put single-headed / single-grained whiteflies of different stages and sexes on the parafilm membrane dripped with 20 μL of extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0), and 0.2 mL The bottom of the PCR tube was fully ground as a homogenizer, and the homogenate was transferred into a 1.5mL centrifuge tube with a micropipette; then the homogenizer was washed twice with 200 μL buffer solution, transferred into the same centrifuge tube, mixed evenly, and 5 μL proteinase K ( 20mg / mL), mix well and place in a water bath at 60°C for 1 hour (mix once in the middle); then bath in boiling water for 8 minutes, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, and mix gently After homogenizing dozens of times, place it on ice for 30 minutes; centrifuge at 4°C, 120...
Embodiment 3
[0061] Example 3: Determination of Primer PPZYF / PPZYR's Detection Threshold of A.
[0062] 1) Preparation of the template DNA of the double-hooked whitefly
[0063] Place female adult whitefly single-headed whitefly on a parafilm membrane dripped with 20 μL extraction buffer (50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 20 mM NaCl, pH 8.0), and use the bottom of a 0.2 mL PCR tube as a homogenate Use a micropipette to transfer the homogenate into a 1.5mL centrifuge tube; then wash the homogenizer twice with 200μL buffer solution, transfer to the same centrifuge tube, mix well, add 5μL proteinase K (20mg / mL), and mix well After homogenization, put it in a water bath at 60°C for 1 hour (mix once in the middle); then bath it in boiling water for 8 minutes, add 220 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, mix it gently for dozens of times, and place on ice Centrifuge at 4°C and 12000r / min for 20min, take the supernatant, add 440μL pre-cooled absolute ethanol, mix gently, and pla...
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