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Specific SS-COII primer of cydia pomonella l. and application thereof

A technology specific to codling moth, applied in the field of agricultural biology, can solve the problems of high homology and difficulty in finding the specific segment of targeted codling moth, achieve simple and fast operation, meet plant quarantine and pest monitoring/ The effect of detection and prevention, simple operation process

Active Publication Date: 2019-09-20
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the analysis shows that the base sequence homology between the above-mentioned different species is relatively high, and it is difficult to find specific segments targeting codling moth

Method used

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  • Specific SS-COII primer of cydia pomonella l. and application thereof
  • Specific SS-COII primer of cydia pomonella l. and application thereof
  • Specific SS-COII primer of cydia pomonella l. and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 codling moth specific primer

[0028] Codling moth specific primers (CPZWF1 / CPZWR1) were designed with the specific segment in the mitochondrial mtDNA COII gene sequence of codling moth as the target sequence, and its sequence is:

[0029] Primer CPZWF1: 5'-ACCATCATTACGTTTATTATAC-3'

[0030] Primer CPZWR1: 5'-AATTGTTCAGGAATGGATG-3'.

[0031] In the present invention, codling moth DNA is used as a template to perform temperature gradient PCR at an annealing temperature of 48-53° C. to determine optimal amplification conditions. For the CPZWF1 / CPZWR1 primer pair, a single band of 262bp can be obtained at each annealing temperature, and the amplified band is the clearest and brightest when the annealing temperature is 51°C, so the annealing temperature is selected to be 51°C.

Embodiment 2

[0032] The specific amplification effect of embodiment 2 primers to codling moth

[0033] Using primers CPZWF1 / CPZWR1 and codling moth DNA as a template, the pear borer, sand fruit borer, peach borer, pear borer, loquat dark spot borer, Lepidoptera sp.1, Lepidoptera sp.2 and head Eight other common species of fruit moths such as Scrobipalpa sp. were used as controls for SS-COII PCR amplification.

[0034] The fruit borer moth larvae (3 instar) collected in the field were confirmed by morphological characteristics and DNA barcode identification, and DNA was extracted and stored at -20°C for future use.

[0035] 5 μL of the PCR product was separated by electrophoresis on a 1.0% (weight / volume) agarose gel containing Gold view, and then judged by the size of the amplified product in a gel imaging system.

[0036] Such as figure 1 As shown, the target band of 262bp has been amplified in the codling moth of 1 swimming lane, and there is no amplified product in the fruit moth of o...

Embodiment 3

[0037] Example 3 Amplification effect of primers CPZWF1 / CPZWR1 on different stages and larval instars of codling moth

[0038] Using primers CPZWF1 / CPZWR1, DNA of codling moth in different stages and larval stages was used as template for PCR amplification.

[0039]DNA was extracted from single head / single codling moths of different developmental stages (eggs, 1st-5th instar larvae, female adults and male adults) (where the 3rd-5th instar larvae took 1 / 2 body length, and adults took the abdomen), Store at -20°C for later use.

[0040] The result is as figure 2 As shown, lanes 1 to 9 all show the target band of 262bp, indicating that codling moth single egg and single newly hatched larvae, 2nd instar larvae, 3rd instar larvae, 4th instar larvae, 5th instar larvae, pupae, codling moth Both female and male adults of the moth can successfully amplify the target gene, therefore, the specific primers of the present invention have a high accuracy rate.

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Abstract

The invention belongs to the field of agricultural biotechnology, and in particular, relates to a specific SS-COII primer of cydia pomonella l. and an application thereof. The specific primer can only have the amplification ability to the cydia pomonella l., the size of the amplified product is 262 bp, and the specific primer has good detection effect on single eggs and adult residual bodies. The primer improves the accuracy of detection of the cydia pomonella l., saves the detection time, and is suitable for promotion in import plant quarantine, domestic plant quarantine and field control in the form of kit.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to codling moth specific SS-COII primers and applications thereof. Background technique [0002] The codling moth Cydia pomonella L., also known as apple tortillaria and apple borer worm, belongs to Lepidoptera Lepidoptera, Tortricidae family Tortricidae, Olethreutinae family Olethreutinae, Cydia genus Cydia, is a worldwide quarantine pest, listed It is one of the most serious fruit-boring pests of pome fruits in the world, and is an important external and internal quarantine object in my country. Codling moth has a wide range of hosts. Therefore, strengthening the monitoring and detection of codling moth is a necessary prerequisite to ensure the healthy development of the fruit industry and dried fruit / nut industry. [0003] Codling moth larvae are borer-boring insects that often occur in secret, especially in the early stages of occurrence, and are difficult t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888
Inventor 张桂芬张正坤王玉生刘万学郭建洋万方浩张毅波张蓉
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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