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Method for detecting and identifying theba pisana through fluorescent quantitative PCR (polymerase chain reaction)

A fluorescence quantitative, pizza tea technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems such as fluorescence quantitative PCR and other problems that have not yet been seen, and achieve the effect of accurate and convenient result judgment, high sensitivity and strong specificity

Active Publication Date: 2017-05-31
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

So far, there is no fluorescent quantitative PCR technology for the detection and identification of pizza tea snails

Method used

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  • Method for detecting and identifying theba pisana through fluorescent quantitative PCR (polymerase chain reaction)
  • Method for detecting and identifying theba pisana through fluorescent quantitative PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Pizza tea snail fluorescent quantitative PCR primer specificity test

[0020] 1. Preparation of materials

[0021] The tested snails are as follows: Pisa tea snail (Spanish specimen), loose big snail; bright big snail; Mediterranean white snail;

[0022] The above-mentioned snails were intercepted in the national port quarantine or obtained through domestic investigation and collection. After being confirmed by the National Mollusc Quarantine and Identification Key Laboratory of the General Administration of Quality Supervision, Inspection and Quarantine, they were stored at -20°C for later use.

[0023] 2. Establishment of fluorescent quantitative PCR method

[0024] 2.1. Design and synthesis of primers: Based on the sequence of the Pisa tea snail ITS2 gene, the primer design soft Primer Express3 was used to assist in the design and analysis of primers. After the specificity was tested by NCBI Blast, the primers were synthesized by Shanghai Sangong Biote...

Embodiment 2

[0034] Example 2: Sensitivity test of fluorescence quantitative PCR detection of pizza tea snail

[0035]The snail DNA stock solution (100 ng / μL) extracted in Example 1 was diluted to 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 There are 8 different concentration gradients of fg / μL, 10 fg / μL and 1fg / μL.

[0036] Fluorescent quantitative PCR amplification reaction system: the total volume is 25 μL, including 2×TaqMan PCR Master Mix 12.5 μL, upstream and downstream primers and probes 1 μL each, 100ng / μL DNA template 2 μL, and the rest with sterilized ddH 2 O make up. After mixing, put it into a fluorescent quantitative PCR amplification instrument for amplification.

[0037] The two-step amplification reaction program of fluorescent quantitative PCR is: 95°C pre-denaturation for 5 minutes; 95°C for 10s, 60°C for 34s, 40 cycles; end the reaction.

[0038] Judgment of fluorescent quantitative PCR results: If a typical amplification curve is observed on the fluorescent ...

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Abstract

The invention provides a method for detecting and identifying theba pisana through fluorescent quantitative PCR (polymerase chain reaction). Specific primers and a probe are used for identifying the molecular features of the theba pisana; an upstream primer of the fluorescent quantitative PCR is TPF: 5'-GCTGGAAAGTGGAGGCCGTA-3'; a downstream primer is TPR: 5'-GCGGCGTCGGTTAAGAGAGA-3'; a fluorescence probe is TPP: 5'-FAM-AGCTGTTCCGGGCACCAAGACGTCACG-BHQ-3'; 5' end marked fluorescence reporter gene is FAM; 3' end marked fluorescence quenching gene is BHQ. The method has the advantages that the theba pisana can be fast and accurately detected and identified; the specificity is high; the sensitivity is high; the consumed time is short; the result judgment is accurate and convenient; the PCR post treatment is not needed; the false positive and the cross contamination can be effectively avoided.

Description

technical field [0001] The invention relates to a method for detecting and identifying pizza tea snails by fluorescent quantitative PCR, which belongs to the technical field of plant quarantine and is suitable for rapid detection and identification of pizza tea snails in entry and exit port quarantine and agricultural production. Background technique [0002] Pizza Tea Snails Theba pisana Belonging to the order Stylommatopgora, large snail family Helicidae, tea snail genus Theba , is a member of the newly promulgated "List of Imported Plant Quarantine Pests of the People's Republic of my country". The snail has an arboreal habit and strong drought resistance. It is very easy to establish populations in new areas such as forest areas and orchards, seriously endangering agricultural and forestry crops and horticultural crops, and the silvery white mucus traces left by the snail after crawling can also seriously reduce the commercial value of crops. value. Pizza Tea Snail ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 周卫川王沛王宇林俊宏林阳武
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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