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Porcine reproductive and respiratory syndrome virus antibody detection method and application thereof

A respiratory syndrome and antibody detection technology, applied in measuring devices, disease diagnosis, biological testing, etc., can solve problems such as non-production, undetectable antibodies, false negatives, etc., to save cumbersome processes and make up for deficiencies and defects Effect

Inactive Publication Date: 2017-08-29
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, we found in the detection of clinical serum samples using IDEXX kits that even individual pigs were immunized with attenuated PRRSV vaccine for 3 weeks and still could not detect antibodies. The inventors analyzed the following two reasons for this, one is that the body is not produce antibodies to PRRSV, and the second is that the existing detection methods cannot detect it, which leads to a significant decline in the accuracy of detection
Antibodies cannot be detected, and the production of antibodies in the body cannot be monitored. It is easy to cause false negatives and immunize here, resulting in misjudgment, formulating unreasonable immunization procedures, causing epidemics, and resulting in economic losses.

Method used

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  • Porcine reproductive and respiratory syndrome virus antibody detection method and application thereof
  • Porcine reproductive and respiratory syndrome virus antibody detection method and application thereof
  • Porcine reproductive and respiratory syndrome virus antibody detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Acquisition of NSP4 Gene

[0035] A. RNA extraction: 200 μL of HP-PRRSV HuN4 vaccine (purchased from Harbin Veken Biotechnology Co., Ltd.) was added with the same volume of Trizol, PRRSV RNA was extracted according to the instructions, the RNA concentration was measured, and then put in a -80°C refrigerator for later use.

[0036] B. Reverse transcription: reverse transcription of the extracted RNA, the reverse transcription system is 20 μL, including: 5× reverse transcriptase Buffer 2 μL, 10mM dNTP MiX 4 μL, RNase inhibitor 1 μL, oligo(dT)18primer 1 μL, AMV Reverse transcriptase 1 μL, RNase-free water 1 μL, template RNA 1 μg; reaction conditions were 42°C, 60 min; 70°C, 5 min.

[0037]C. PCR amplification of the target fragment: use the cDNA synthesized by reverse transcription as a template for PCR amplification. The reaction system was 50 μL: 1 μL each of the upstream and downstream primers, 25 μL of TaKaRa Premix Taq, 2 μL of cDNA, and water to 50 μL. Re...

Embodiment 2

[0043] Embodiment 2 NSP4 expression and purification

[0044] A. Expression and identification of NSP4 protein: Transform the recombinant expression plasmid pET28a-NSP4 into the competent cells of the expression strain Trasseta (DE3), use the kana resistance to screen the recombinant transformants, pick a single colony and activate it in LB medium overnight. The ratio of 1% was transferred to fresh LB medium containing kana-resistant and cultured until the OD600 was about 0.8, adding IPTG with a final concentration of 1 mM, 225 rpm, 30°C shaking culture for 5 hours. The cells were collected by centrifugation at 10,000 g at 4°C for 10 min, resuspended in PBS, ultrasonically disrupted, and centrifuged at 12,000 rpm at 4°C for 15 min, and the supernatant and precipitate were collected for SDS-PAGE identification. The results showed that the target protein was highly expressed, with a size of about 26KDa, and most of the target protein was located in the lysed supernatant.

[004...

Embodiment 3E

[0047] Embodiment 3ELISA condition optimization

[0048] Each step in the ELISA process is optimized, and the optimized result is:

[0049] A. Antigen coating amount: 50-1000ng of purified NSP4 was used as the coating protein, and it was found that the P / N value was the highest when coating 200ng;

[0050] B. Coating conditions: Different coating buffers and action time were detected, and it was found that the coating buffer was a phosphate buffer with pH=7.2, and its 1L solution contained 0.356g NaH 2 PO 4 , 2.772 g Na 2 HPO 4 12H 2 O, 8.5g NaCl, the highest P / N value when coated overnight;

[0051] C. Blocking conditions: with the blocking solution containing 2.5% skimmed milk powder with a pH of 7.2 PBS'T buffer, the P / N value is the highest when incubated at 37°C for 1 hour;

[0052] D. Serum sample dilution: the PBS'T+volume concentration that is 7.2 is 2.5% skimmed milk powder and is diluted by 1:40, and the P / N value is the highest when adding 100 μ l;

[0053] E...

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Abstract

The invention relates to the field of animal virus antibody diagnosis detection, and in particular provides a porcine reproductive and respiratory syndrome virus (PRRSV) antibody detection method and application thereof. The method comprises the following steps: firstly, cloning a non-structure protein NSP4 of a PRRSV HUN4 vaccine strain into a pET-28a (+) prokaryotic expression vector, performing low-temperature induction soluble expression, purifying by utilizing an His marker, and by taking purified protein as coating antigen, establishing an indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method; confirming a critical value as 0.406, detecting 1274 cases of clinical pig serum by utilizing the ELISA method, detecting by utilizing an American IDEXX kit at the same time, and comparing results, wherein the positive rate of the 1274 cases of clinical pig serum is 71.59%, and the positive rate agreement rate is up to 92%. The detection method provided by the invention is capable of effectively implementing deficiency of the IDEXX kit and can be used as a common PRRSV antibody detection method.

Description

technical field [0001] The invention relates to the field of animal virus antibody detection methods, and provides a porcine reproductive and respiratory syndrome virus antibody detection method and application thereof. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease caused by PRRS virus (PRRSV). Contact infectious diseases, mainly characterized by loss of appetite, persistent fever, abortion, premature birth, mummified fetus and weak litter in pregnant sows, as well as high mortality and respiratory diseases in suckling piglets and finishing pigs. Some recovered sows showed symptoms of repeated infertility and atypical estrus. At present, the disease has become one of the major infectious diseases that endanger the world's swine industry. The disease not only lacks effective drugs for treatment, but also the existing vaccines cannot effectively control PRRSV infection, resulting in continuous outbreaks of P...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/561G01N33/535
CPCG01N33/68G01N33/535G01N33/561G01N33/6893G01N2800/26
Inventor 肖一红刘思当商营利曹胜亮蔡新娜
Owner SHANDONG AGRICULTURAL UNIVERSITY
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