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Kit for simultaneously detecting six common porcine viruses in Fukuotou raw material and product of Fukuotou raw material

A kit and virus technology, applied in the field of biochips, can solve problems that have not been reported, and achieve the effects of improving detection sensitivity, detection efficiency and accuracy, and ensuring specificity and accuracy

Pending Publication Date: 2022-04-12
台州市药品检验研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0031] After searching, there has been no report on the gene chip that uses PCR products and membrane gene chip hybridization to simultaneously detect six common viruses

Method used

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  • Kit for simultaneously detecting six common porcine viruses in Fukuotou raw material and product of Fukuotou raw material
  • Kit for simultaneously detecting six common porcine viruses in Fukuotou raw material and product of Fukuotou raw material
  • Kit for simultaneously detecting six common porcine viruses in Fukuotou raw material and product of Fukuotou raw material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1 kit preparation process

[0075] 1. Bioinformatics analysis and specific primer screening

[0076] Search different porcine virus genome sequences, perform systematic sequence comparison through DNA MAN, NCBI BLAST, DNA STAR and other software, select conservative and specific genes or sequences as detection objects, and then use primer6 or oligo7 and other software to design multiple pairs of specific primers , and further comparative analysis was carried out in the primers of the software, and primers with small interactions between primers were selected for synthesis and testing. .

[0077] 2. Sample preparation

[0078] For the extraction of total nucleic acid (including DNA and RNA) of the sample, the viral genome DNA / RNA extraction kit (or other extraction methods or kits with equivalent efficacy) can be used to extract the total nucleic acid, and Concentration quantitative analysis was performed.

[0079] 3. PCR primer specificity test

[0080] ...

Embodiment 2

[0139] According to the instructions of Tiangen Biochemical Technology (Beijing) Co., Ltd. Viral DNA / RNA Extraction Kit (Product No.: DP315), the total nucleic acids of 6 kinds of porcine virus standard samples were extracted respectively. The concentration and quality of the extracted total nucleic acid were measured by an ultra-micro spectrophotometer (NanoDrop2000), and then PCR samples were added to test the amplification efficiency and specificity of each primer.

[0140] The reaction system of PCR amplification is as follows:

[0141] 2×RT-Multiplex PCR Master Mix: 10 μL;

[0142] Alternative primers for each target (10μM): 0.5μL

[0143] Alternative primers for each target (10μM): 0.5μL

[0144] Extracted sample total nucleic acid: 2-5 μL;

[0145] wxya 2 O: Make up to a total volume of 20 μL;

[0146] After the amplification, gel electrophoresis was performed to detect the amplified target band. Primers with good specificity and high amplification efficiency (that ...

Embodiment 3

[0148] Preparation of multiplex reaction system: use primer pairs 1-7 to prepare multiplex RT-PCR primer Mix, and the final concentration of each primer is 10 μM. Add 0.4 μL of prepared Primer Mix to each 20 μL reaction system, so the final working concentration of each primer in the reaction system is 0.2 μM.

[0149] Nucleic acid extraction: According to the instructions of Tiangen Biochemical Technology (Beijing) Co., Ltd. Viral DNA / RNA Extraction Kit (Cat. No.: DP315), the total nucleic acids of the six porcine virus standard samples were extracted respectively. The concentration of the extracted total nucleic acid was measured with an ultramicro spectrophotometer (NanoDrop2000), and the above nucleic acid was gradiently diluted to an intermediate concentration (about 1.0E+04copies / μL) with enzyme-free water for PCR loading.

[0150] The reaction system of PCR amplification is as follows:

[0151] 2×RT-Multiplex PCR Mix (including Primer Mix): 10.4μL;

[0152] Extracted ...

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Abstract

The invention provides a kit for detecting six common porcine viruses in a Fukuo raw material and a product thereof. The kit comprises a gene membrane chip, a hybridization reagent and an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) reaction system, the gene membrane chip comprises a nylon membrane substrate, and oligonucleotide probes, contrast probes and blank dot coatings which are distributed on the nylon membrane substrate in an array manner; the RT-PCR reaction reagent comprises a combination of seven primer pairs. Multiple PCR is combined with a reverse dot blot hybridization technology, seven specific primer pairs are utilized, seven specific gene segments can be amplified at the same time, and the specificity and accuracy of a detection result are further ensured through hybridization of PCR products and probes on a membrane chip; compared with common qualitative PCR and fluorescent quantitative PCR, the detection efficiency and accuracy are greatly improved.

Description

technical field [0001] The invention relates to a biochip of a gene microarray, in particular to a kit for simultaneously detecting six kinds of pig common viruses in Fuketuo raw materials and its products, belonging to the technical field of biochips and detection reagents. Background technique [0002] Spleen aminopeptide oral freeze-dried powder (Fu Ketuo) is a freeze-dried product made of polypeptides and nucleotides extracted from fresh pig spleen through dialysis, and mannitol is added as an excipient. The drug is an immunomodulator, mainly used for the treatment of low cellular immune function, immunodeficiency and autoimmune dysfunction diseases (recurrent respiratory tract infection, bronchitis, pneumonia, asthma, severe herpes zoster and psoriasis, etc.); for malignant tumors Improve the patient's quality of life after radiotherapy, chemotherapy and postoperative, and reduce the incidence of colds, fevers or other infections caused by various reasons. The main aud...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6806C12Q1/686C12Q1/6837C12N15/11C12R1/93
CPCY02A50/30
Inventor 洪亮潘映秋王福云
Owner 台州市药品检验研究院
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