Method of synchronous distinguishing newcastle disease virus and vaccine virus and identifying virulence and genotype

A newcastle disease virus and vaccine virus technology, applied in the field of automated analysis software, can solve the problems of small number of verification samples, insufficient theoretical foundation, and difficult application

Inactive Publication Date: 2007-03-14

HUZHOU TEACHERS COLLEGE

0 Cites 5 Cited by

AI-Extracted Technical Summary

Problems solved by technology

However, its theoretical basis is based on the difference of a small number of nucleotides in the primer region between a small number of virulent and attenuated nucleic acid sequences, which ...

Abstract

The present invention discloses molecular biological method and automatic analysis software for distinguishing wild virus strain and vaccine virus strain of new castle disease virus (NDV), and identifying the toxicity and genotype simultaneously. The NDV is RT-PCR amplified with one pair of specific degenerate primers to obtain one multipurpose NDV segment for direct sequencing of the PCR product. One automatic analysis software is composed for comparing the homology of the sequence to the standard vaccine sequence so as to distinguish wild virus strain and vaccine virus strain, analyze the toxicity by means of the F gene cracking site and judge the genotype through developing tree analysis. The present invention has short detecting period, reliable detection result and capacity of obtaining several detection results simultaneously, and is suitable for use in fowl product inspector and quarantiner.

Application Domain

Microbiological testing/measurement

Technology Topic

Direct sequencingDegenerate primer +11

Image

Examples

Experimental program(1)

Example Embodiment

[0116] Example 1-Isolation and detection of Newcastle disease strains

[0117] 1. Isolation and identification of viruses.

[0118] According to the method of "Diagnostic Laboratory and Vaccine Standard Manual" (edited by the International Bureau of Epidemiology, 1996), 51 strains of Newcastle disease virus were isolated from geese, pigeons, chickens, and ostriches.

[0119] 2. Extraction of viral nucleic acid RNA

[0120] (1) Take 100 μl of allantoic fluid to be tested.

[0121] (2) Add 900μl TRIzol reagent (Gibco-BRL company), mix well, and room temperature for 5 minutes;

[0122] (3) Add 0.2ml or an appropriate amount of chloroform, shake and mix, and then stand at room temperature for 3 minutes;

[0123] (4) Centrifuge at 12000g, 4°C for 15 minutes;

[0124] (5) Carefully transfer the supernatant to a new 1.5ml centrifuge tube;

[0125] (6) Add 0.5ml or an appropriate amount of isopropanol, shake and mix well, and place at room temperature for 10 minutes;

[0126] (7) Centrifuge at 12000g, 4°C for 10 minutes;

[0127] (8) Discard the supernatant and add 0.5ml of 75% ethanol to wash once;

[0128] (9) 12000g, 4℃, centrifuge for 5min;

[0129] (10) Discard the supernatant, and dry the pellet at 37°C for 10 minutes;

[0130] (11) Add 20μl RNase free ddH 2 O dissolves.

[0131] 2. RT-PCR amplification and direct sequencing of PCR products:

[0132] among them: P+ : 5’ATG GGC ICC AIA ICT TCT ACC 3’

[0133] P- : 5’CAT TGG TTG CIG CAA TGC TCT C3’

[0134] A. RT reaction:

[0135] (1) Add 2μl P+ (10pmol/μl) in 10μl of nucleic acid;

[0136] (2) Incubate at 100°C for 3 minutes;

[0137] (3) Quickly insert into ice for 5 minutes;

[0138] (4) Add 4μl of 5X AMV RTase buffer, 2μl of dNTP mixture (10mM), 1μl of Ribonuclease inhibitor (TaKaRa company), and 1μl of AMV RTase (5U/μl) (TaKaRa company). Stir gently

[0139] (5) 10min at room temperature;

[0140] (6) Move to 42℃ constant temperature water bath for 1 hour;

[0141] (7) All the reaction solution is used for PCR reaction.

[0142] B. PCR reaction:

[0143] (8) Join

[0144] 10X PCR Buffer(Mg 2+ free) 20μl;

[0145] MgCl 2 (25mM) 12μl;

[0146] dNTP(2.5mM) 16μl;

[0147] P+ (10pmol/μl) 8μl;

[0148] P- (10pmol/μl) 8μl;

[0149] rTaq(5U/μl) 1μl; (TaKaRa company)

[0150] RT product 20μl;

[0151] ddH 2 O 115μl

[0152] Total 200μl

[0153] (9) After mixing, 100 μl aliquot into 2 thin-walled tubes; PCR reaction.

[0154] (10) PCR reaction conditions:

[0155] 94℃ 4mins

[0156]

[0157] 72℃ 10mins

[0158] (11) Take 15μl gel electrophoresis detection (the product is 480bp in size, Figure 12)

[0159] C. Direct sequencing of PCR products

[0160] (12) Take 170μl PCR product (the remaining 15μl-20°C reserve) and send it to the biotechnology company for tapping and recovery, and then perform direct sequencing of the PCR product (the target band is located at 480bp, usingP- As a sequencing primer).

[0161] 3. Use Newcastle Disease Virus to analyze the sequence:

[0162] (1) E-mail to receive the company's sequencing results;

[0163] (2) Start the Newcastle disease virus tyrant software and enter the main analysis interface.

[0164] (3) Click the "load sequence" button to enter the sequence;

[0165] (4) Click the "Detect" button, the software first asks to select the location to save the results, after confirming, the software will automatically analyze and output the results.

[0166] 4. The results are shown in Table 10. Among the 51 isolated strains, the vaccine accounted for 58.8%; the suspected vaccine was 3.9%; the wild virus was 37.3%. The vaccine was mainly La Sota, and no VH and Newcastle disease strain I were detected; wild virus The main ones are genotype VII, genotype VI and genotype II.

[0167] Vaccine

PUM

Description & Claims & Application Information

We can also present the details of the Description, Claims and Application information to help users get a comprehensive understanding of the technical details of the patent, such as background art, summary of invention, brief description of drawings, description of embodiments, and other original content. On the other hand, users can also determine the specific scope of protection of the technology through the list of claims; as well as understand the changes in the life cycle of the technology with the presentation of the patent timeline. Login to view more.