Cassava mealybug specific ss-coi primer pair and rapid PCR detection method and kit
A cassava mealybug and detection kit technology, applied in the field of molecular biology, to achieve the effect of simple operation process, high application value, simple and fast operation
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Embodiment 1
[0028]Example 1: Amplification effect of primers PMZWMEF1 / PMZWMER1 on cassava mealybug
[0029] 1) Preparation of mealybug template DNA
[0030] Place the single-headed mealybug on the parafilm membrane dripped with 20 μL extraction buffer (10 mM Tris-HCl, 50 mM EDTA, 0.5% SDS, 100 mM NaCl, 0.2% β-mercaptoethanol, pH 8.0), and use a 0.2 mL PCR tube The bottom was used as a homogenizer to grind thoroughly, and the homogenate was transferred into a 1.5mL centrifuge tube with a micropipette; then the homogenizer and Parafilm membrane were washed twice with 180 μL buffer solution, transferred to the same centrifuge tube, mixed evenly, and 5 μL proteinase K ( 20mg / mL), mix thoroughly and place in a water bath at 65°C for 40 minutes (mixed twice in the middle); then bath in boiling water for 8 minutes, add 200 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, and mix gently After dozens of times, place it on ice for 30 minutes; centrifuge at 4°C and 7500r / min for 10 minutes, tak...
Embodiment 2
[0044] Example 2: Amplification effect of primers PMZWMEF1 / PMZWMER1 on cassava mealybugs of different stages, ages, residues and host plants
[0045] 1) Preparation of cassava mealybug template DNA
[0046] Single / single-grained cassava mealybugs of different stages (adult, egg, nymph) and instars (1st, 2nd, 3rd instar) from pumpkin, and single female adults (from pumpkin and cassava, respectively) and its head, thorax, abdomen, and feet (all from pumpkin) were placed in drops of 20 μL extraction buffer (10 mM Tris-HCl, 50 mM EDTA, 0.5% SDS, 100 mM NaCl, 0.2% β-mercaptoethanol, pH 8.0) Use the bottom of a 0.2mL PCR tube as a homogenizer to grind fully, and transfer the homogenate into a 1.5mL centrifuge tube with a micropipette; then wash the homogenizer and Parafilm membrane twice with 180μL buffer, and transfer it to the same Centrifuge the tube, mix well, add 5 μL proteinase K (20mg / mL), mix well, and put it in a water bath at 65°C for 40 minutes (mixing twice in the middl...
Embodiment 3
[0058] Embodiment 3: Primer PMZWMEF1 / PMZWMER1 is to the determination of cassava mealybug detection threshold
[0059] 1) Preparation of cassava mealybug template DNA
[0060] A single female adult of Pycnotica cassava was placed on a parafilm membrane dripped with 20 μL extraction buffer (10 mM Tris-HCl, 50 mM EDTA, 0.5% SDS, 100 mM NaCl, 0.2% β-mercaptoethanol, 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 and the Parafilm membrane were washed twice with 180 μL buffer solution, transferred to the same centrifuge tube, mixed, and 5 μL Proteinase K (20mg / mL), mix well and place in a water bath at 65°C for 40 minutes (mixed twice in the middle); then bath in boiling water for 8 minutes, add 200 μL of chloroform / isoamyl alcohol (V:V=24:1) extract, After gently mixing dozens of times, place on ice for 30 minutes; centrifuge at 4°C, 7500r / m...
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