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Duplex nanometer PCR detection method for identifying CCoVI and CCoVII

A detection method and nanotechnology, applied in the field of genetic engineering, can solve problems such as unsuitable canine viruses, achieve good sensitivity, high sensitivity and specificity, and avoid the effect of non-specific amplification

Active Publication Date: 2018-10-19
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is not yet applicable to the identification and detection of canine viruses, especially CCoVI and CCoVII

Method used

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  • Duplex nanometer PCR detection method for identifying CCoVI and CCoVII
  • Duplex nanometer PCR detection method for identifying CCoVI and CCoVII
  • Duplex nanometer PCR detection method for identifying CCoVI and CCoVII

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: CCoVI and CCoVII RT-PCR amplification of target genes

[0039] 1. Primer design:

[0040] The primers CCoVII-F and CCoVII-R of the present invention are used to amplify the specific sequence of CCoVII, and the size of the amplified fragment is 239bp; the primers CCoVII-F and CCoVII-R are used to amplify the specific sequence of CCoVII, and the size of the amplified fragment is 105bp. Primers were synthesized by Beijing Huada Biotechnology Co., Ltd. The primer sequences are detailed below:

[0041] CCoVI-F: 5'-GTGCTTCCTCTTGAAGGTACA-3' (SEQ ID NO.1);

[0042] CCoVI-R: 5'-TCTGTTGAGTAATCACCAGCT-3' (SEQ ID NO.2);

[0043] CCoVII-F: 5'-TAGTGCATTAGGAAGAAGCT-3' (SEQ ID NO.3);

[0044] CCoVII-R: 5'-GCAATTTTGAACCCTTC-3' (SEQ ID NO. 4).

[0045] The nucleotide sequences of specific target fragments obtained after amplification are shown in SEQ ID NO.5 (CCoVI) and SEQ ID NO.6 (CCoVII).

[0046] 2. Establishment of common RT-PCR detection methods for CCoVI and CCoV...

Embodiment 2

[0049] Embodiment 2: The establishment of CCoVI and CCoVII nanometer RT-PCR (nanoRT-PCR) detection method

[0050] 1. Optimization of annealing temperature for CCoVI and CCoVII nanoRT-PCR:

[0051] Using the recombinant plasmids pMD-CCoVI and pMD-CCoVII as templates, the annealing temperature was set at 46°C to 53°C using a temperature gradient PCR instrument, and the annealing temperature was optimized by increasing by 1°C. The reaction system was as follows: Taq DNA polymerase (5U / μL) (GRED, Shandong, China), 0.5 μL; 2×nano PCR Buffer (GRED, Shandong, China), 10 μL; CCoVI-F, CCoVI-R, CCoVII-F and 1 μL each for CCoVII-R; 1.0 μL each for pMD-CCoVII and pMD-CCoVII; add ddH2O to 20 μL. The reaction conditions were as follows: 95°C, 3min; 35 cycles: 95°C, 30s, 46°C-53°C (1°C in order), 30s, 72°C, 30s; 72°C, 10min.

[0052] CCoVI and CCoVⅡ nanoRT-PCR was optimized by annealing temperature (46℃~53℃), and the results of agarose gel electrophoresis are shown in figure 2 . It can...

Embodiment 3

[0064] Example 3: Detection and verification of clinical samples

[0065] 1. Collection and processing of clinical samples:

[0066] A total of 60 dog feces samples were obtained from an animal hospital in Beijing. A small amount of dog feces was taken and diluted 1:4 with 0.01mol / LPH7.6PBS. After centrifugation at 12000r / min for 5min, the supernatant was taken and applied with Axyprep Body FluidViral DNA Genomic RNA was extracted using the / RNA Miniprep kit. Using this RNA as a template, cDNA was synthesized according to the instructions of the M-MLV reverse transcription kit, and the cDNA of the obtained sample was stored at -20°C until testing.

[0067] 2. Detection and verification:

[0068] CCoVI and CCoVII were detected on cDNA of 60 clinical samples by using nanoRT-PCR established in the present invention. The test results showed that the positive rate of CCoVII was 11.67%, the positive rate of CCoVII was 48.33%, and the mixed infection rate of the two was 8.33% (see T...

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Abstract

The invention discloses a nanoRT-PCR detection method for identifying CCoVI and CCoVII. Through the optimization on the primers, annealing temperature and primer concentration in a nanoRT-PCR reactionsystem, the effective amplification of respective target strips of the CCoVI and the CCoVII can be obtained; meanwhile, the non-specific amplification between primers can be avoided. The lowest nucleic acid detection quantity of the nanoRT-PCR detection method is 100 times higher than that of an ordinary RT-PCR method; the products with the identical size with the expected segment size and the completely correct sequence are obtained through nanoRT-PCR amplification. The nanoRT-PCR method for identifying CCoVI and CCoVII provided by the invention has ultra-high sensitivity and specificity, belongs to an efficient detection measure, can be applied to the epidemic investigation of canine coronavirus diseases; meanwhile, the foundation can be laid for the identification and prevention and control of the CCoV; great practical significance and values are realized.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a dual nanometer RT-PCR detection method for distinguishing CCoVI and CCoVII and an application thereof. Background technique [0002] Canine coronavirus (CCoV) is one of the main pathogens causing canine gastroenteritis. It generally causes non-fatal mild diarrhea in young dogs, showing the characteristics of high morbidity and low mortality. When combined with other intestinal Mixed infection with pathogenic pathogens can lead to high mortality of sick animals. In recent years, with the successive reports of highly pathogenic mutant strains of CCoV in European countries, the disease has once again attracted widespread attention [1-2]. [0003] CCoV is a single-stranded positive-sense RNA virus belonging to the order Nestoviridae, the family Alphacoronaviridae, and the genus Alphacoronavirus [3]. In recent years, different genotypes of CCoV have been detected, wh...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2563/137C12Q2563/155
Inventor 崔尚金秦彤
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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