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Multiple nested PCR (polymerase chain reaction) detection primer, kit and method for Neospora caninum, Brucella abortus and infectious bovine rhinotracheitis virus

A technology of rhinotracheitis virus and Brucella, applied in the field of molecular genetics, can solve the problems that the existence of antibodies cannot fully reflect the pathogen, the specificity and sensitivity of identification need to be improved, time-consuming and labor-intensive, and achieve a simple and reasonable system composition, Reaction program optimization, the effect of short time consumption

Inactive Publication Date: 2017-08-04
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For Neosporosis (currently a very important disease that causes abortion in dairy cows), there are only imported commercial serum antibody detection kits, which are expensive, but the presence of antibodies in animals cannot fully reflect whether there must be presence of antibodies in animals. pathogen
In addition, these three diseases need to be tested separately, that is, it costs a lot of money to buy three kinds of kits, and it is time-consuming and laborious
[0005] The Chinese invention patent with the publication number CN103409543A discloses a multiplex PCR detection kit for detecting bovine neosporidiosis and toxoplasmosis internal standard. PCR identification method, its identification specificity and sensitivity need to be improved

Method used

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  • Multiple nested PCR (polymerase chain reaction) detection primer, kit and method for Neospora caninum, Brucella abortus and infectious bovine rhinotracheitis virus
  • Multiple nested PCR (polymerase chain reaction) detection primer, kit and method for Neospora caninum, Brucella abortus and infectious bovine rhinotracheitis virus
  • Multiple nested PCR (polymerase chain reaction) detection primer, kit and method for Neospora caninum, Brucella abortus and infectious bovine rhinotracheitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Neospora, Brucella and bovine infectious rhinotracheitis virus multiplex nested PCR detection primers in this embodiment include: outer primer set:

[0082] Primer NC EX 1: 5'-CTTTTCAACCCTCAACCTTT-3';

[0083] Primer NC EX 2: 5'-TGACACCCAATCATACTGCTC-3';

[0084] Primer BA EX 1: 5'-CTACGGAATAACTCAGGGAAAC-3';

[0085] Primer BA EX 2: 5'-AACCCAACATCTCACGACAC-3';

[0086] Primer IBRV EX 1: 5'-ACGGACCTGGTGGACAAGAA-3';

[0087] Primer IBRV EX 2: 5'-GTGCGTGCCGTTGTAGCG-3';

[0088] Internal primer set:

[0089] Primer NC IN 3: 5'-TGAGTTGTATCGCCTTCTT-3';

[0090] Primer NC IN 4: 5'-ATTCACATTGCGTTTCG-3';

[0091] Primer BA IN 3: 5'-GGTAAAGGCTCACCAAGGC-3';

[0092] Primer BA IN 4: 5'-CAGGCGGAATGTTTAATGC-3';

[0093] Primer IBRV IN 3: 5'-AGGTGGTGGCCTTTGACC-3';

[0094] Primer IBRV IN 4: 5'-TCGTCTCGCAGCATTTCGTC-3'.

Embodiment 2

[0096] In this embodiment, Neospora, Brucella and bovine infectious rhinotracheitis virus multiple nested PCR detection kits include: outer primer set, inner primer set, positive control template, negative control template, TranStart Taq DNA polymerase, 10×TranStart Taq Buffer, dNTPs.

[0097] The outer primer set is:

[0098] Primer NC EX 1: 5'-CTTTTCAACCCTCAACCTTT-3';

[0099] Primer NC EX 2: 5'-TGACACCCAATCATACTGCTC-3';

[0100] Primer BA EX 1: 5'-CTACGGAATAACTCAGGGAAAC-3';

[0101] Primer BA EX 2: 5'-AACCCAACATCTCACGACAC-3';

[0102] Primer IBRV EX 1: 5'-ACGGACCTGGTGGACAAGAA-3';

[0103] Primer IBRV EX 2: 5'-GTGCGTGCCGTTGTAGCG-3';

[0104] The inner primer set is:

[0105] Primer NC IN 3: 5'-TGAGTTGTATCGCCTTCTT-3';

[0106] Primer NC IN 4: 5'-ATTCACATTGCGTTTCG-3';

[0107] Primer BA IN 3: 5'-GGTAAAGGCTCACCAAGGC-3';

[0108] Primer BA IN 4: 5'-CAGGCGGAATGTTTAATGC-3';

[0109] Primer IBRV IN 3: 5'-AGGTGGTGGCCTTTGACC-3';

[0110] Primer IBRV IN 4: 5'-TCGTCTCGCAGCAT...

Embodiment 3

[0113] The multiple nested PCR detection method of Neospora, Brucella and bovine infectious rhinotracheitis virus in the present embodiment comprises the steps:

[0114] 1) extract the DNA of the sample after mixing the brain, liver and lung tissue of the fetal bovine;

[0115] 2) Use the DNA obtained in step 1) as a template, add the outer primer set, and perform the first round of PCR amplification; the system for the first round of PCR amplification is 25 μl, including: 2.5 μl of 10×TranStart Taq Buffer (purchased From Beijing Quanshijin Company), 1.5mmol / L of dNTPs, 0.6μmol / L of each primer in the outer primer set, 1.5U of TranStart Taq DNA polymerase, 1μl of template, and water as the balance; the reaction procedure is: 95 ℃ pre-denaturation for 3 minutes; 95℃ for 30s, 53℃ for 1min, 72℃ for 1min, 35 cycles; 72℃ for 10min; get the first round of amplification products;

[0116] 3) Use the first-round amplification product as a template, add the internal primer set, and perf...

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Abstract

The invention relates to multiple nested PCR (polymerase chain reaction) detection primer, kit and method for Neospora caninum, Brucella abortus and infectious bovine rhinotracheitis virus, and belongs to the technical field of molecular genetics. The multiple nested PCR detection kit comprises inner and outer primer pairs for detecting Neospora caninum ITS1, Brucella abortus 16S rRNA and infectious bovine rhinotracheitis virus gB gene; by using the kit, it is possible to synchronously detect 3 pathogens causing abortion of dairy cows. The multiple nested PCR detection method has very high sensitivity and specificity; infection conditions of 3 abortive pathogens can be detected at a time, the detection speed is high, and the cost is greatly saved. In the detection method, components of various PCR systems are optimized such that system composition is simple and reasonable; reaction procedures of each phase are optimized such that amplification results are easy to distinguish and amplification takes short time.

Description

technical field [0001] The invention relates to multiple nested PCR detection primers, a kit and a detection method for Neospora, Brucella and bovine infectious rhinotracheitis virus, and belongs to the technical field of molecular genetics. Background technique [0002] Abortion is a common disease during pregnancy of dairy cows. Abortion of dairy cows leads to extended calving intervals, increases the cost of feed for raising cattle, affects the acquisition of high milk yield and the expansion of heifers, and causes serious economic losses to the dairy farming industry. According to statistics, the average economic loss per abortion case is 640 US dollars. It is generally believed that the annual abortion rate of cattle farms is 3% to 5%, which is a normal phenomenon, but according to recent surveys, the abortion rate of domestic dairy cows has reached 10% to 25%. Diagnosis and analysis of the causes of abortion are of great significance to the prevention and control of a...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12N15/11C12R1/93C12R1/01
CPCC12Q1/6848C12Q1/6888C12Q1/689C12Q1/701C12Q2531/113C12Q2537/143
Inventor 钱伟锋王天奇闫文朝位治国张旻韩利方李静吕超超
Owner HENAN UNIV OF SCI & TECH
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