Specific SS-COI (species-specific-COI) primer based PCR rapid detection method for Frankliniella intonsa
A detection method and a specific technology, which are applied in the field of molecular biology, can solve the problems that the SS-COI method has not yet been applied in the detection of thrips, and achieve the effects of simple and rapid operation, strong specificity and high accuracy.
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Embodiment 1
[0041] Example 1, using primers Fi63-4F / Fi63-4R to amplify flower thrips
[0042] (1) Preparation of the genome of Thrips florensis
[0043] Collect flower thrips in the cotton field, put single-head flower thrips into a 1.5mL centrifuge tube and grind it, and use OMEGA InsectGenomic DNA Kit extraction kit (OMEGA Company, USA) to extract single-head thrips according to the operating procedures attached to the kit. Genomic DNA of Thrips analis. Measure the concentration of the DNA solution (10.1 ng / μL) on a DNA concentration analyzer (Nanodrop, ND2000C, American Thermoelectric), store it at -20°C for later use, and draw 1 μL of the solution as a DNA template during PCR amplification.
[0044] (2) synthetically detect the specific SS-COI primer of Thrips floridae, the sequence is
[0045] Specific Forward Primer Fi63-4F for Thrips analis: 5'-CGCTAAGTATTTTAATTCGG-3'(SEQ ID No.1)
[0046] Specific reverse primer Fi63-4R for Thrips analis: 5'-AGTGGCACTAGTCAGTTTCC-3' (SEQ ID No.2...
Embodiment 2
[0052] Example 2, the specific amplification effect of primers Fi63-4F / Fi63-4R on Thrips florida
[0053] Utilize primers Fi63-4F / Fi63-4R, use Thrips floridae DNA as a template, and 32 other insects in the same domain as Thrips floridae (see Table 1) as controls for SS-COI PCR amplification, the results are as follows figure 1 shown. Only the 150bp target band was amplified in the 1 and 2 lanes corresponding to Thrips florida, and there were no corresponding bands in the other lanes, indicating that the SS-COI primers designed according to the mitochondrial DNA COI gene of Thrips florida had strong specificity.
[0054] Table 1. Insect species involved in primer-specific assays
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Embodiment 3
[0057] Example 3, Primer Fi63-4F / Fi63-4R Determination of Minimum Detection Amount of Thrips floridae
[0058] According to the method in Example 1, the genomic DNA of Thrips unicephalus was extracted, and then the original template concentration of 10.1 ng / μL was diluted by 4 times until no band was detected, and 1 μL was used as a template for PCR detection, and PCR reaction was carried out. Its reaction system is the same as above.
[0059] Using primers Fi63-4F / Fi63-4R to measure the minimum detection amount, the minimum detection concentration of DNA of Thrips floridus is 0.158ng / μL, the results are as follows figure 2 shown.
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