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Liquid-phase chip method used for detecting infectious laryngotracheitis virus

A technology of laryngotracheitis virus and liquid phase chip, which is applied in the field of molecular biology, can solve the problems of time-consuming, labor-intensive sensitivity, inability to perform high-throughput detection, and inability to meet high-throughput quarantine and other problems

Inactive Publication Date: 2013-01-02
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although classic methods such as virus isolation, serological diagnosis and enzyme-linked immunosorbent assay are time-consuming, labor-intensive and have poor sensitivity, they cannot detect subclinical infection, and subclinical infection of infectious laryngotracheitis plays an important role
Although PCR technology has the characteristics of fast, specific, sensitive and easy to operate, it has been widely used in the detection and differential diagnosis of infectious diseases of livestock and poultry, but it cannot be used for high-throughput detection, cannot meet the requirements of high-throughput quarantine, and is not conducive to For the quarantine of a large number of customs clearance samples and the emergency screening of a large number of samples during an outbreak, it is necessary to establish a simple, fast and accurate screening and inspection technology for infectious laryngotracheitis to meet the requirements of daily detection

Method used

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  • Liquid-phase chip method used for detecting infectious laryngotracheitis virus
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  • Liquid-phase chip method used for detecting infectious laryngotracheitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0047] Example 2 Extraction and amplification of infectious laryngotracheitis virus DNA

[0048] 1. The extraction of ILT virus DNA was carried out with reference to the instructions of the commercial kit (DNAeasy Blood and Tissue kit) purchased from QIAGEN Company, and the specific steps were as follows:

[0049] (1) Take 180 μL of viral allantoic fluid in a 1.5 mL EP tube, add 20 μL of proteinase K, mix well, and centrifuge briefly.

[0050] (2) Add 200 μL of BufferAL to the sample, vortex to mix, centrifuge briefly, and incubate at 56°C for 10 min.

[0051] (3) After the white precipitate is dissolved, add 200 μL of absolute ethanol to the sample, vortex to mix, and centrifuge briefly.

[0052] (4) Install the DNeasy Mini Spin Column in the kit on the collection tube (2mL), transfer the solution in step (3) to the DNeasy Mini Spin Column, centrifuge at ≥6000g (8000rpm) for 1min at room temperature, discard Go to collection tube and filtrate.

[0053] (5) Put the DNeasy M...

Embodiment 3

[0063] Example 3 Establishment of liquid phase chip detection method

[0064] 1. Coupling of probes and microspheres

[0065] Suspend the fluorescently encoded microspheres on a vortex at a speed of 2800r / min, and then ultrasonicate the microspheres at room temperature for 30s on an ultrasonic cleaner. Take 50μL (containing 6.25×10 5 pcs) microspheres into a centrifuge tube and centrifuge at 12000g for 2min. Discard the supernatant, resuspend the microspheres with 10 μL of 0.1M MES (2-morpholineethanesulfonic acid) at pH 4.5, and vortex at 2800 r / min for 30 seconds on a vortexer to disperse the microspheres. Add 2 μL of probe ILTV-P (0.1 nmol / μL) in Table 1 in Example 1. Prepare fresh EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] solution (10 mg / mL), add 2 μL of EDC solution to the microspheres, and mix well. Incubate for 30 min at room temperature in the dark. Prepare fresh EDC solution (10mg / mL) again, add 2μL EDC solution to the microspheres, and mix well. Incu...

Embodiment 4

[0076] Embodiment 4 Characteristic detection of ILTV liquid phase chip method of the present invention

[0077] 1. Specificity test results

[0078] The liquid chip detection method established in this study was used to detect common avian infectious bronchitis virus (IBV) AV10, avian influenza virus subtypes H5N1, H7N1, H9N2 and Newcastle disease virus (NDV) purchased from the China Veterinary Drug Administration. Viral nucleic acid was detected, and each sample was repeated twice. The results are shown in Table 4. The fluorescence values ​​of the liquid chip detection results of pathogens such as IBV, NDV, AIV H5N1, AIV H9N2, and AIV H7N1 were not significantly different from those of the blank control, which was less than 3 times the fluorescence value of the blank control, indicating that the detection results were all Negative, while the fluorescence values ​​of ILTV were all greater than 200, and greater than 3 times the fluorescence value of the blank control. It show...

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Abstract

The invention provides a liquid-phase chip method used for detecting infectious laryngotracheitis virus (ILTV). Through comparison and analysis upon ILTV TK genome sequence, conserved regions are found, and specific primers and a probe of the ILTV are designed. The nucleotide sequences are respectively represented by SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. The primers above are used for carrying out PCR amplification upon ILTV DNA, and the probe is coupled with microspheres. A PCR amplification product and the probe coupled on fluorescent microspheres are subjected to hybridization. Through the detection upon fluorescence values, the liquid-phase chip method used for detecting ILTV is established. The detection method provided by the invention has the advantages of accurate detection, high sensitivity, and high specificity. The method is simple and fast.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting infectious laryngotracheitis virus by means of a liquid phase chip. Background technique [0002] Infectious laryngotracheitis (ILT) is an acute, contact upper respiratory infectious disease caused by the herpesviridae α-herpesvirinae infectious laryngotracheitis virus (ILTV), characterized by dyspnea and coughing up blood. The main features are mucus-like mucus, edema and hemorrhage of laryngeal and tracheal mucosa followed by erosion. Severe economic losses are caused by decreased egg production and flock mortality, and the disease can infect chickens of all ages and breeds. The disease usually breaks out suddenly and spreads quickly among chickens. The infection rate is about 90% to 100%, and the fatality rate is 5% to 70%, generally between 10% and 20%. May et al. reported the disease for the first time in the United States in 1925, and now it has spr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C40B40/06G01N21/64
Inventor 王慧煜韩雪清林祥梅梅琳李志辉
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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