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Preparation method of shrimp white spot syndrome virus disease nucleic acid sample and shrimp white spot syndrome virus detection method

A white spot syndrome and sample preparation technology is applied in the field of detection of shrimp white spot syndrome virus and nucleic acid sample preparation of shrimp white spot syndrome virus disease, which can solve problems such as extinction of production, loss of farmers, and explosive death of farmed shrimp, and achieve The effect of simple operation, less sample and low cost

Inactive Publication Date: 2010-09-22
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] White spot syndrome virus disease is a severe viral infectious disease. Its pathogen is white spot syndrome virus (WSSV). WSSV is a virus with strong infectivity and high lethality. %-100%, and its host domain is very extensive, it has become a serious threat to marine and freshwater cultured prawns or crayfish. It may even cause the entire pond to be out of production, causing heavy losses to the majority of farmers.

Method used

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  • Preparation method of shrimp white spot syndrome virus disease nucleic acid sample and shrimp white spot syndrome virus detection method
  • Preparation method of shrimp white spot syndrome virus disease nucleic acid sample and shrimp white spot syndrome virus detection method
  • Preparation method of shrimp white spot syndrome virus disease nucleic acid sample and shrimp white spot syndrome virus detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Using different template preparation methods to prepare templates for amplification and perform LAMP amplification, and compare the amplification results.

[0034] The specific operation process is as follows:

[0035] (1) Extraction of template DNA:

[0036] The first type of method, the classic phenol / chloroform extraction method: Take Procambarus clarkii confirmed to be naturally infected with WSSV, kill them, cut them into pieces, take 0.1g of fresh tissue, add 100μl of TE, ① add 500μl of saturated phenol, and mix well 10min, then centrifuge at 10000rpm for 10min, take the water phase, ② repeat ①, then add 500μl phenol / chloroform (1:1), mix well, centrifuge at 10000rpm for 10min, take the water phase, ③add 500μl chloroform and mix, then centrifuge at 10000rpm 10 min, take the water phase, ④ add 1 / 10 volume of 3M sodium acetate, and 2 times the total volume of 95% ethanol to mix, centrifuge at 10,000 rpm for 10 min, discard the supernatant, wash the preci...

Embodiment 2

[0042] Example 2: Buffer III was used to prepare templates by quick cooking method to amplify specific regions in WSSV ORF 234 and WSSV ORF 131 respectively.

[0043] The specific operation process is as follows:

[0044] (1) Extraction of template DNA: In Example 1, the template prepared by adding buffer III to the WSSV diseased shrimp tissue of Macrobrachium rosenbergii after quick cooking was used as mother liquor, and then after 10-fold serial gradient dilution, used as template respectively WSSV ORF 234 and WSSV 131 were amplified in LAMP.

[0045] (2) The primers for LAMP amplification of WSSV ORF 234 are FIP-234, BIP-234, F 3 -234,B 3 -234; the primers for amplifying WSSV ORF 131 are: FIP-131 see SEQ ID No.5 (5'-CCA GTG CAC TAT TCC CAT TAG AAG GTT TTA CCA CTC CCT TCATTA TTG TC-3'), BIP-131 see SEQ ID No.6 (5'-CCT TTT GCC CCT TCAGCT GA TTT TCT GAG CCA GGT GTT CTG-3'), F3-131 see SEQ ID No.7 (5'-TGT GGA TGA TTA TCC TGT GTT-3' ), B3-131 see SEQ ID No.8 (5'-TCT CTC TGG ...

Embodiment 3

[0051] Example 3: Comparison of the sensitivity of detecting WSSV with LAMP, one-step PCR and nested PCR

[0052] (1) Preparation of template DNA: Mince 0.1 g of Procambarus clarkii WSSV diseased shrimp tissue, add 100 μl of buffer III, put in a water bath at 100°C for 5 min, and then centrifuge at 5000 rpm for 5 min, then use the supernatant as a template for LAMP reaction.

[0053] (2) LAMP amplifies WSSV ORF 234, the method is the same as in Example 1.

[0054] (3) One-step PCR of WSSV ORF 234 to detect WSSV: the two primer sequences of WSSV ORF 234 one-step PCR are respectively P1 see SEQ ID No.9 (5'-CCG AAT TCA CCA TGG AGTATA TAG GGG-3'), P2 see SEQ ID No. 10 (5'-CGA AGC TTG ATA CAG TGACCG TCC CTG-3').

[0055] 25μL one-step PCR reaction system includes 2.5μL 10×PCR buffer, 2.0μL MgCl 2 (25mmol / L), each of 0.25μL of P1 and P2, 0.5μL of the prepared template, 0.5μL of dNTPs (2.5mmol / L), 1U of Taq DNA polymerase. The reaction conditions were pre-denaturation at 94°C for ...

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Abstract

The invention belongs to the field of virus detection in the biological technology and particularly relates to a preparation method of a nucleic acid sample for diagnosing a shrimp white spot syndrome virus disease and a method for detecting a shrimp white spot syndrome virus by using the nucleic acid sample subjected to LAMP (Loop-mediated Isothermal Amplification). The method for detecting a shrimp white spot syndrome virus comprises the following steps: taking killed fresh shrimp tissue, slightly shearing the killed fresh shrimp tissue and adding the sheared shrimp tissue into buffer solution III; quickly boiling and centrifuging to obtain supernate as a template for amplifying nucleic acid; after carrying out the LAMP, adding SYBR Green I into the loop-mediated isothermal amplification(LAMP) diluted by thousand folds or ten thousand folds; and judging a detection result by observing whether to exist an excited green fluorescent result through naked eyes under the ultraviolet excitation. The invention has the characteristics of high sensitivity, high specificity, simple and convenient operation, short time consumption, fewer required samples, low cost and the like.

Description

technical field [0001] The invention belongs to the field of virus detection in biotechnology, and in particular relates to a method for preparing a nucleic acid sample of shrimp white spot syndrome virus disease and a method for detecting shrimp white spot syndrome virus after the nucleic acid sample is amplified by LAMP. Background technique [0002] White spot syndrome virus disease is a severe viral infectious disease. Its pathogen is white spot syndrome virus (WSSV). WSSV is a virus with strong infectivity and high lethality. %-100%, and its host domain is very extensive, it has become a serious threat to marine and freshwater cultured prawns or crayfish. Even can cause whole pond to be out of production, cause heavy loss to numerous raisers. At present, WSSV has spread to major shrimp farming countries and regions in Asia, and has spread to North America. Among all the shrimp viruses that have been reported, the virus is the most virulent and the most harmful. Penaeu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/24C12Q1/70C12Q1/68
Inventor 薛仁宇贡成良曹广力魏育红朱越雄沈卫德周小燕
Owner SUZHOU UNIV
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