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Primer probe combination and kit for detecting pathogen nucleic acids of human herpes virus and application of primer probe combination and kit

A human herpes virus and detection kit technology, applied in the field of microorganism and nucleic acid genome detection, can solve the problems of poor DNA polymerase activity, improper temperature, and invalid indication results, etc.

Active Publication Date: 2021-07-16
冯志山
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the result shows a positive CIC, it indicates that the result is reliable and authentic, while when the CIC is negative, it indicates that the result is invalid, and the possible reasons are operator error, improper temperature, incorrect RAA reaction system mixture, poor DNA polymerase activity or inhibition in the sample matrix substance, resulting in a false negative result

Method used

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  • Primer probe combination and kit for detecting pathogen nucleic acids of human herpes virus and application of primer probe combination and kit
  • Primer probe combination and kit for detecting pathogen nucleic acids of human herpes virus and application of primer probe combination and kit
  • Primer probe combination and kit for detecting pathogen nucleic acids of human herpes virus and application of primer probe combination and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The construction of embodiment 1 recombinant plasmid and internal reference plasmid

[0047] The genome sequences of CMV and Human B-globin (HHB) were downloaded from the NCBI website, and the highly conserved sequences were screened and compared with Vector11.1 software.

[0048] The CIC sequence replaces the position of the target probe with the rosette sequence in the selected CMV genome sequence, and the CIC probe sequence is a complementary rosette sequence. The position of the CIC probe sequence change and the CIC-RAA reaction diagram of the whole real-time internal control control are as follows figure 1 As shown, the NCIC-RAA response graph is shown as figure 2 shown.

[0049] Entrust a professional company to synthesize and return the target fragment.

Embodiment 2

[0050] Design and synthesis of embodiment 2 primers and probes

[0051] According to the principles of RAA primer and probe design, primers and probes were manually designed for CMV UL123 gene and HHB gene. The lengths of primers and probes ranged from 30-35bp and 46-52bp, respectively. The probes consist of upstream oligonucleotides carrying 5'-FAM (target gene probe) and 5'-HEX (CIC probe), respectively connected to adjacent downstream oligonucleotides via THF spacers. The 3' end of the downstream oligonucleotide has a C3' spacer (polymerase extension blocking group), and the specificity of the primers and probes is analyzed and evaluated with Oligo7.0 software and Primer BLAST software of NCBI. The sequences of the probes are shown in Table 1.

[0052] Table 1

[0053]

[0054]

[0055] All primers and probes were synthesized by a professional company, the primers were purified by PAGE, and the probes were purified by HPLC. The three probes use double-labeled prob...

Embodiment 3

[0056] Example 3 Optimization of RAA

[0057] 1. Optimization of CIC-RAA: In order to determine the optimal concentration and template concentration of CIC, and ensure that the normal amplification of CIC does not affect the target gene amplification curve, different CIC template concentrations (500, 200, 100 and 50 copies / μL ) in the presence of , we optimized the concentration of the CIC template by detecting standard plasmids 1, 5, and 10 copies.

[0058] 1) The reaction system includes: 25 μL buffer, 2.1 μL forward primer (as shown in SEQ ID No.4, 10 μmol / L), 2.1 μL reverse primer (as shown in SEQ ID No.5, 10 μmol / L), 0.6 μL target gene probe (as shown in SEQ ID No.6, 10 μmol / L), 0.6 μL CIC probe (as shown in SEQ ID No.7, 10 μmol / L), 1 μL CIC plasmid (respectively 500, 200 , 100, 50 copies / μL). Adjustable volumes of nuclease-free water and template.

[0059] CMV CIC Sequence 5-3:

[0060] GATAACCAAGCCTGAGGTTATCAATGTCATGAAGCGCCGCATTGAGGAGATCTGCATGAAGGTCTTTGCCCAGTACATTCT...

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Abstract

The invention relates to the field of detection of microorganisms and nucleic acid genomes, in particular to a primer combination and a probe for detecting nucleic acids of a clinically common human herpes virus (cytomegalovirus) by using an isothermal nucleic acid amplification technology. The primer combination and the probe provided by the invention are good in specificity and high in sensitivity.

Description

technical field [0001] The invention relates to the field of detection of microorganisms and nucleic acid genomes, in particular to a combination of primers and probes for detection of pathogenic nucleic acids of human herpesviruses, a kit and applications thereof. Background technique [0002] Cytomegalovirus (CMV) is a double-stranded DNA virus in the human herpesvirus family, which can cause primary and recurrent diseases and seriously endanger human life and health, especially in immunocompromised and organ transplant patients . CMV infection is highly related to human congenital diseases, which can easily lead to neonatal growth retardation, nerve deafness, congenital deafness and other diseases. CMV, human herpesviruses, and other related viruses can cause respiratory disease, neurological infections, and infectious mononucleosis, among other clinical disorders. The clinical symptoms of diseases caused by these pathogens are very similar. [0003] At present, the pa...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/705C12Q1/6844
Inventor 冯志山高源赵梦川李贵霞帖彦清
Owner 冯志山
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