Processing method and detecting method for sample of nosema bombycis naegeli in graine by utilizing PCR (Polymerase Chain Reaction) method

A treatment method and detection method technology, applied in the field of microbial detection, can solve the problems of time-consuming and other problems, and achieve the effect of simple operation and low cost

Inactive Publication Date: 2013-02-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention provides a method for processing silkworm egg samples, which solves the problem that the existing PCR metho

Method used

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  • Processing method and detecting method for sample of nosema bombycis naegeli in graine by utilizing PCR (Polymerase Chain Reaction) method
  • Processing method and detecting method for sample of nosema bombycis naegeli in graine by utilizing PCR (Polymerase Chain Reaction) method
  • Processing method and detecting method for sample of nosema bombycis naegeli in graine by utilizing PCR (Polymerase Chain Reaction) method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Detection of Silkworm Eggs Infected with Microsoma Bombyx mori

[0035] (1) Primer design

[0036] For the small subunit ribosomal RNA gene (N bombycis SSU rRNAFJ854546.1) of Bombyx mori, specific primers were designed, and the length of the amplified fragment was 408bp, wherein:

[0037] Forward primer (NB-SSU599F): 5'-ATAAATCGGAGGGCAAAT-3';

[0038] Reverse primer (NB-SSU599R): 5'-TAAGCCGCACAATCCAC-3'.

[0039] (2) Sample preparation and processing

[0040] Preparation of the second-generation silkworm eggs infected with M. silkworm: routinely raise silkworm eggs, and after the 5th instar silkworms are raised, carry out different spore concentrations of M. 3 、10 4 、10 5 、10 6 and 10 7 moths / mL) were licked and eaten within 4 hours, followed by routine feeding, stacking, eclosion, mating (the male moths used for mating were healthy individuals) and oviposition, and the eggs laid after mating were the second-generation silkworms. Eggs, pickling the sil...

Embodiment 2

[0057] Example 2 Detection of Healthy Silkworm Eggs Mixed with Bombyx mori Microspores Spores

[0058] (1) Primer design

[0059] Primer design was the same as step (1) in Example 1. .

[0060] (2) Sample processing

[0061] Put healthy silkworm eggs (eggs immediately soaked in pickling, refrigerated eggs soaked in pickling, polymorphic silkworm eggs or naturally aged silkworm eggs) in an incubator at 25°C and a relative humidity of 80% for 10 days , take silkworm eggs that are green or turn blue, put them into a PCR tube, add 5 μL sample pretreatment solution (containing 1mol / L sodium hydroxide and 5mol / L betaine), cover them, place them at 25°C for more than 30 minutes, and use 50 μL Crush the silkworm eggs with the tip of a pipette, and add 1.0 μL of M. silkworm spore liquid. The concentration of M. silkworm spores is 10 5 、10 7 and 10 9 To each / mL, add 5.0 μL of 1.0mol / L hydrochloric acid, cover and place for PCR amplification, and use it up on the same day.

[0062...

Embodiment 3

[0067] Example 3 Detection of Bombyx mori microspores in dead eggs

[0068] (1) Primer design

[0069] Primer design was the same as step (1) in Example 1.

[0070] (2) Sample preparation and processing

[0071] The silkworm eggs were reared conventionally. After the silkworms were raised at the 5th instar, they were licked according to the dose of 100 μL Bombyx mori spore liquid per silkworm. The concentration of Bombyx mori spores was 10 4 Each / mL, eat up within 4 hours, then carry out routine feeding, stacking, eclosion, mating (the male moth used for mating is a healthy individual) and oviposition, and the eggs laid after mating are the second generation silkworm eggs.

[0072] Immediately dip the second-generation silkworm eggs in acid and routinely accelerate greening until they turn green, pick out 10 dead eggs, and the egg prints of the dead eggs are triangular, put them into PCR tubes, put one in each PCR tube, and add 5 μL of sample pretreatment solution (containi...

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Abstract

The invention discloses a processing method for a sample of a nosema bombycis naegeli in a graine by utilizing a PCR (Polymerase Chain Reaction) method. The processing method comprises the steps as follows: adding a graine sample to a pretreatment solution for at least 30min; mashing the graine and adjusting the pH value of the solution to neutral so as to obtain a PCR template, wherein the pretreatment solution is alkaline and contains 1 to 10mol/L of glycine betaine. In addition, the invention further provides a method for detecting the nosema bombycis naegeli in the graine by utilizing the sample processing method. The detecting method provided by the invention has the advantages of simplicity in operation and low in cost, and is capable of shortening the time of the whole detection process by 40%, thereby being suitable for detection of common nosema bombycis naegeli and specifically suitable for quarantine inspection of the nosema bombycis naegeli in the graine (silkworm eggs, silkworm chrysalis and moths) with greater sample capacity.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and relates to a sample processing method and a detection method for detecting silkworm micronoids in silkworm eggs by a PCR method. Background technique [0002] Nosema bombycis Naegeli 1857 is the pathogen of silkworm micronosis. Bombyx mori is a protozoan. When it parasitizes the silkworm, it appears most in the form of spores. The spores are single-celled structures, usually oval, surrounded by a 0.5 micron thick spore wall. The spore wall is very tough and can withstand weak acids And alkali, strong resistance to the outside world. Bombyx mori microparticle disease is a systemic chronic silkworm disease with a long course of disease, and there are different symptoms and lesions in each developmental stage of silkworm. Quarantine object. [0003] From 1865 to 1970, French scientist Louis Pasteur conducted a series of studies on silkworm microparticle disease, and found that...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 鲁兴萌蔡顺风李明乾马焕艳何永强李光才
Owner ZHEJIANG UNIV
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