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Fluorescent PCR detection reagent for identifying bovine gene source in heparin, preparation method and application

A heparin and gene technology, applied in the field of biological detection reagents, can solve the problems of unsuitable heparin medium bovine gene detection, unfavorable heparin medium bovine gene test, unfavorable large-scale testing, etc., and achieve high specificity, intuitive results, and easy operation. Effect

Active Publication Date: 2015-10-28
BEIJING POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this system is not suitable for the detection of bovine genes in heparin. The main reasons can be summarized as the following three points: 1) The research results of many people show that heparin polysaccharides have an inhibitory effect on PCR amplification enzymes and cannot be directly used in real- Time PCR experiment must be done after certain treatment; 2) The method in this patent is relatively complicated, and multiple PCR is not conducive to the detection of bovine genes in heparin; 3) In addition, the reaction system of this method is 50 μl, which consumes a lot of reagents , which greatly increases the detection cost, which is not conducive to the realization of large-scale detection in enterprises

Method used

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  • Fluorescent PCR detection reagent for identifying bovine gene source in heparin, preparation method and application
  • Fluorescent PCR detection reagent for identifying bovine gene source in heparin, preparation method and application
  • Fluorescent PCR detection reagent for identifying bovine gene source in heparin, preparation method and application

Examples

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Embodiment 1

[0054] A biological detection reagent for identifying the source of bovine genes in heparin, comprising a pair of primers and a TaqMan probe, the pair of primers are respectively the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, the The TaqMan probe is the nucleotide sequence shown in SEQ ID NO.3.

[0055] Further, the 5' end of the TaqMan probe SEQ ID NO.3 is connected with a fluorescent reporter group, and the 3' end is connected with a quenching group.

[0056] Further, the fluorescent reporter group connected to the 5' end is 6-carboxyfluorescein (FAM), and the quencher group connected to the 3' end is 6-carboxytetramethylnordamine (TAMRA).

[0057] A preparation method of a biological detection reagent for identifying bovine gene sources in heparin, the preparation method comprising the following steps:

[0058] 1) Select the specific and conserved conserved sequence fragment of the Bov-A2 ​​gene in the bovine mitochondrial gene as the target, and the amplif...

Embodiment 2

[0085] This embodiment is a preferred solution of embodiment 1, which is the detection of bovine genes in crude heparin.

[0086] 1. Design primers and probes

[0087] In this example, the specific and conserved conserved sequence fragment of the Bov-A2 ​​gene in the bovine mitochondrial gene is selected as the target, and by collecting a large number of bovine mitochondrial gene sequences reported in GenBank and the mitochondrial gene sequences of various other ruminants and non-ruminants, For sequence analysis and comparison, use PrimerExpress3.0 software and PrimerSelect software in DNAStar to design and synthesize primers and probes to be tested. After conducting the optimal pairing screening experiment on the designed and synthesized primers and probes, the candidate primers and probes are initially determined, after a large number of reaction conditions optimization, comparative tests and verification tests, and after a large number of sample detection application evalua...

Embodiment 3

[0119] This embodiment is the preferred solution of embodiment 1, which is the detection of bovine gene in high-quality heparin.

[0120] 1. Design primers and probes

[0121] In this example, the specific and conserved conserved sequence fragment of the Bov-A2 ​​gene in the bovine mitochondrial gene is selected as the target, and by collecting a large number of bovine mitochondrial gene sequences reported in GenBank and the mitochondrial gene sequences of various other ruminants and non-ruminants, For sequence analysis and comparison, primers and probes to be tested were designed and synthesized using Primer Express3.0 software and PrimerSelect software in DNAStar. After conducting the optimal pairing screening experiment on the designed and synthesized primers and probes, the candidate primers and probes are initially determined, after a large number of reaction conditions optimization, comparative tests and verification tests, and after a large number of sample detection ap...

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Abstract

The invention discloses a fluorescent PCR detection reagent for identifying cattle gene source in heparin, a preparation method and application, provides a detection method for detecting cattle composition source in a heparin crude finished product, a real-time fluorescence quantification PCR primer and a probe by performing sequencing and comparison on cattle gene, and the length of an amplification target fragment for cattle composition detection is 92 bp. The invention also provides a kit for performing quantitative determination on cattle gene. The detection method and the detection kit have the advantages of accurate detection, high sensitivity, strong specificity and the like, and have good specimen detection capability.

Description

technical field [0001] The invention relates to a biological detection reagent capable of identifying the source of bovine genes in heparin, as well as a preparation method and application of the reagent. Background technique [0002] Heparin is an anticoagulant, which is a polymer composed of two polysaccharides connected alternately, and has anticoagulant effects both in vivo and in vitro. Clinically, it is mainly used for thromboembolic diseases, myocardial infarction, cardiovascular surgery, cardiac catheterization, extracorporeal circulation, hemodialysis, etc. With the progress of pharmacology and clinical medicine, the application of heparin has been expanding. [0003] In 2010, the export volume of my country's heparin and its salt hit a record high, and it has become my country's largest export commodity of western medicine. In 2010, my country's heparin and its salts were exported to 51 countries and regions, with a high degree of export concentration. The top fi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2561/101C12Q2563/107
Inventor 李晔全爽金丽华
Owner BEIJING POLYTECHNIC
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