Porcine circovirus type 2 LAMP detection primer group and kit

A porcine circovirus and detection kit technology, applied in the biological field, can solve the problems of undiagnosed clinical diagnosis, delayed detection time, complicated and cumbersome virus isolation and identification, etc.

Pending Publication Date: 2021-09-03
LONGYAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Diagnosis methods for PCV2 include clinical diagnosis, virus isolation and identification, enzyme-linked immunosorbent assay (ELISA), common PCR and fluorescent quantitative PCR, etc. Among them: clinical diagnosis cannot be confirmed, virus isolation and identification are too complicated and cumbersome, and ELISA detection can only be performe

Method used

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  • Porcine circovirus type 2 LAMP detection primer group and kit
  • Porcine circovirus type 2 LAMP detection primer group and kit
  • Porcine circovirus type 2 LAMP detection primer group and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Design of PCV2 Virus LAMP Detection Properties.

[0032] According to the nucleic acid sequence of the published PCV2 virus, a LAMP detection primer group for PCV2 viruses is designed, and the specific nucleotide sequence is as follows:

[0033] SEQ ID NO.1 (PCV2-F3): 5'-gctgtgagtaccttgtgggag-3 '

[0034] SEQ ID NO.2 (PCV2-B3): 5'-acaaccacttctcaccatg-3 '

[0035] SEQ ID NO.3 (PCV2-FIP): 5'-TgcatttcccgctCCCCCCCTTTTGCAGAGCAGCCCCCCTGTAAC-3 '

[0036] SEQ ID NO.4 (PCV2-BIP): 5'-AtgtacacgtcattgtggggTTCTAGGTGTTTCCAGTATGTGTGTTTCCAGTATGTG-3 '

[0037] SEQ ID NO.5 (PCV2-LF): 5'-agcccgggaaatttctg-3 '

[0038]SEQ ID NO.6 (PCV2-LB): 5'-gtgtggcaaaaagcaaatgg-3 '

[0039] Preparation of positive control plasmid at PCV2 virus LAMP: Synthesis of PCV2 viral LAMP amplification of the corresponding DNA fragment and attached to the plasmid PGH, the plasmid named PGH-PCV2, used as a positive control at the time of PCV2 virus LAMP detection, The specific sequence is shown in SEQ ID NO.7.

[0040]...

Embodiment 2

[0048] PCV2 virus LAMP detection kit sensitivity test.

[0049] PGH-PCV2 plasmid was diluted with a series of dilutions, dilution from 4 ^ (- 1) -4 ^ (- 9), and then the dilution of each dilution was detected using this kit. Such as figure 2 Indicated.

[0050] After 60 min at 61 ° C, from 4 ^ (- 1) -4 ^ (- 5) dilution and 4 ^ (- 7) dilution showed dark blue, 4 ^ (- 6) dilution is a reaction tube dark blue The other reaction tube is a purple red, 4 ^ (- 8) -4 ^ (- 9) dilution is purple. This result shows that the highest sensitivity of this kit can reach 4 ^ (- 7) dilution, and the number of PGH-PCV2 plasmid copies containing the dilution is 18.1 copies.

[0051] The above results show that the PCV2 viral LAMP detection kit of the present invention is good, and its maximum detection sensitivity can reach 18.1 copies of target molecules.

Embodiment 3

[0053] Detection of PCV2 Virus LAMP Detection Kit on Clinical Samples.

[0054] 10 clinical samples were detected by the PCV2 viral LAMP detection kit of the present invention while comparing with the normal PCR detection results. Such as image 3 Indicated.

[0055] The results showed that in 10 clinical samples, 4 positive samples were detected in the PCV2 virus LAMP kit, and the remaining 6 were negative samples, which were consistent with the normal PCR test results. This result illustrates the PCV2 virus LAMP detection kit of the present invention to be used for PCV2 virus detection of clinical samples.

[0056] In summary, the PCV2 virus LAMP detection kit of the present invention has a series of advantages of high sensitivity, good specificity, short reaction time, and simple results visualization, and simple supporting instrument. The kit is not only suitable for large breeding units and high-end laboratories, but also for medium and small farm units and general laboratorie...

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Abstract

The invention discloses a porcine circovirus type 2 LAMP (Loop-Mediated Isothermal Amplification) detection primer group and a kit, the porcine circovirus type 2 LAMP detection primer group comprises six primers, and the specific nucleotide sequences of the six primers are respectively as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The porcine circovirus type 2 LAMP detection kit comprises the primer group, and also comprises the following components: Bst DNA polymerase, dNTP, MgSO4, Betaine, a 10X LAMP reaction buffer solution, HNB, a positive control plasmid and the like. The kit disclosed by the invention has a series of advantages of high sensitivity, good specificity, short reaction time, result visualization, simple matched instruments and the like, and is not only suitable for large breeding units and high-end laboratories, but also suitable for vast small and medium breeding units and common laboratories.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and specifically, the porcine ring virus type 2 LAMP detection primer group and kit. Background technique [0002] Porcine Circovirus (PCV) belongs to the circoviridae ring virus, which is a single-stranded DNA virus, a phenocapstick. Depending on its discovery, the difference in pathogenic and nucleic acid sequences can be divided into type 2, ie PCV1 and PCV2. PCV1 is not pathogenic, while PCV2 has a pathogenic. [0003] Porcine Circovirus Type 2 (PCV2) is a DNA virus with a single negative chain cyclic phenademic membrane, and its genome is 1.7KB. The PCV2 infection mainly causes the POSTWEANING MULTISISTEMIC WANDROME (PMWS), which can be combined with many pathogens, causing immunosuppression in pigs. [0004] Since the first outbreak of PCV2 in the Canadian pigs in 1991, it is widely popular among the pigs around the world. PCV2 Relevance Disease, PCVAD is a complex, multi-factor disease, in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 俞国华杨小燕戴爱玲范克伟刘建奎黄翠琴杨守深尹会方李晓华王鑫包银莉
Owner LONGYAN UNIV
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