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Hepatitis a virus detecting method in food

A hepatitis A virus and detection method technology, applied in the field of bioengineering, can solve the problems of high missed detection rate, complicated operation, low sensitivity, etc., and achieve the effect of ensuring safety, high sensitivity and specificity

Inactive Publication Date: 2009-10-07
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection methods of hepatitis A virus mainly include the following types, but all of them have deficiencies: (1) cell culture method: this method is to use the characteristics of hepatitis A virus to reproduce and replicate in host cells, and to cultivate cells infected by hepatitis A virus in vitro. After 30 to 60 days, observe the cell lesions. This method takes a long time and the operation is too complicated, so it is not suitable for the detection of hepatitis A virus in food; (2) ELISA method: mainly through the detection of anti-HAV-IgG antibodies in serum, but currently ELISA The sensitivity and specificity of the method detection are all low, and the missed detection rate is high; (3) RT-PCR method: this method mainly detects by the amplification of the target gene fragment of hepatitis A virus, but the current RT-PCR method sensitivity Relatively low, the detection limit of hepatitis A virus is generally only about 10 3.0 TCID 50
However, the currently disclosed methods for detecting hepatitis A virus in food using fluorescent quantitative PCR technology are still not perfect.

Method used

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  • Hepatitis a virus detecting method in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Design and synthesis of primers

[0025] According to the sequence of Hepatitis A in Genbank database, Primer Premier 5.0 software was used to design primers, and the primer sequences are shown in Table 1.

[0026] Table 1 Hepatitis A virus primer sequence

[0027] Primer

[0028] The primers were synthesized by the solid-phase phosphoramidite triester method and entrusted to Shanghai Yingjun Biotechnology Co., Ltd. to complete.

Embodiment 2

[0029] Embodiment two: the processing of standard sample

[0030] Dissolve the hepatitis A live attenuated vaccine powder with 1.0ml of water for injection, and the amount of live virus contained in it is 10 7.0 TCID 50 , stored at 4°C for later use.

[0031] Dilute the above-mentioned hepatitis A live attenuated vaccine solution into 10 5.0 TCID 50 、10 4.0 TCID 50 、10 3.0 TCID 50 、10 2.0 TCID 50 、10 1.0 TCID 50 、10 0 TCID 50 A total of 6 concentration gradients were stored at 4°C for future use.

Embodiment 3

[0032] Embodiment three: the processing of sample to be tested

[0033] 1. Processing of shellfish samples

[0034]Dissect and remove the intestinal tissue from shellfish, take 1.5g sample and add 15ml Tris_HCL buffer (pH=7.4, 0.05mol / L), homogenate on ice; put the homogenate into a 50ml centrifuge tube, and incubate at 37°C for 30min ;Centrifuge at 5,000g for 20min at 4°C, collect the supernatant into a new tube; add PEG-8000 at a concentration of 10% of the supernatant, add NaCl at a final concentration of 0.4mol / L, and place overnight at 4°C; The next day, centrifuge at 10,000g for 5min at 4°C, and take the precipitate; add 10ml of Tris_HCL buffer solution to the precipitate, add an equal amount of chloroform after fully dissolving, shake at room temperature for 30min; centrifuge at 2,000g for 30min at 4°C, and take the upper layer of water Phase, stored at -20°C for future inspection.

[0035] 2. Processing of meat samples

[0036] Directly weigh 1.5g of meat products (...

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Abstract

The present invention discloses an hepatitis a virus detecting method in food, the detecting method including following steps : 1) processing of sample under test; 2) RNA extraction of sample under test; 3) RNA reverse transcription; 4) SYBR Green I Real-time PCR reaction; 5) judgement of detected results: when cyclic threshold value of the sample under test is less than 33, judging as hepatitis a virus positive; when cyclic threshold value of the sample under test is more than 35, judging as hepatitis a virus negative; when cyclic threshold value of the sample under test is less than 35 and more than 33, treating as suspicious sample, if the renewed detected results is still in the range, then judging as hepatitis a virus negative. The detecting method of the invention is provided with advantages of rapid, high sensitivity and specificity and quantitative. judging as hepatitis a virus positive.

Description

technical field [0001] The invention relates to bioengineering, more specifically, the invention relates to a method for detecting hepatitis A virus in food. Background technique [0002] Viral hepatitis A is a common disease that seriously endangers the health of the broad masses of the people. It is caused by hepatitis A virus infection. Hepatitis A virus (HAV) is a non-enveloped single-stranded positive-sense RNA virus belonging to the Hepadnavirus genus of the Picornaviridae family. Hepatitis A virus is distributed all over the world, and my country is one of the high-incidence areas, and its incidence rate is very high. Hepatitis A virus mainly causes human infection through the fecal-oral gastrointestinal route, and aquatic products, vegetables, water and other foods can act as transmission media due to contamination, causing the occurrence and prevalence of hepatitis A. Worldwide, about 7% of all cases of infectious hepatitis A are attributable to eating raw or impr...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 高东微莫雪梅
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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