Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method, oligonucleotide and kit for HBV genotype detection

An oligonucleotide and genotyping technology, applied in the biological sciences and biological fields, can solve the problems of poor specificity, missed detection, high cost, etc., and achieve the effects of eliminating false positive results, avoiding cross-reactions, and preventing false negatives

Pending Publication Date: 2021-10-26
LEADWAY HK
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, because SYBR GreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman probe method, and its specificity must be judged by observing the melting curve; while the cost of the double-probe hybridization method is more expensive
[0007] At present, most reagents for HBV genotyping in the domestic market are mainly for detecting types B and C. Although there are a few products that can detect types D, the existing HBV genotyping reagents cannot effectively distinguish between types C, D and C / D recombinant type, so in the actual detection process, it often happens that HBV C / D recombinant type cannot be detected, or the C / D recombinant type is regarded as C type or D type, which is not conducive to accurate guidance of patients' medication, nor Conducive to monitoring the patient's disease progression and treatment prognosis
In addition, considering that there are still a small number of patients carrying genotypes other than A, B, C, D and C / D recombinant types, although some detection products can detect A, B and C types, and even D types, they cannot. Detect if other HBV genotypes are present, which can lead to missed detection

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method, oligonucleotide and kit for HBV genotype detection
  • Method, oligonucleotide and kit for HBV genotype detection
  • Method, oligonucleotide and kit for HBV genotype detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: sample DNA extraction

[0046] In Aikang Biotechnology (Hangzhou) Co., Ltd. On the NES-32 nucleic acid purification instrument, the DNA of clinical samples was extracted using the reagents in the nucleic acid (DNA) extraction kit (magnetic bead method) of Aikang Biotechnology (Hangzhou) Co., Ltd.

[0047] 1) Add the reagent solutions shown in Table 2 to the 96-well plate in advance (this step can be omitted if pre-loaded plate reagents are used):

[0048] Table 2. Nucleic acid extraction solutions

[0049]

[0050] 2) Add 20 μl of proteinase K to the first column and the seventh column of the 96-well plate in sequence; then add 200 μl of the sample to be extracted;

[0051] 3) Edit the program on the NES-32 nucleic acid purifier according to the steps shown in Table 3 and run:

[0052] Table 3. Nucleic acid extraction steps

[0053]

[0054] 4) After the automatic extraction, transfer the eluted products of columns 6 and 12 to a 1.5ml centrifuge tu...

Embodiment 2

[0055] Embodiment 2: fluorescent PCR reaction steps

[0056] Prepare PCR reaction solution 1 for detecting HBV virus types A, B and HBV DNA, 40 μl of detection system. The detailed formula of PCR reaction solution 1 is as follows:

[0057] PCR reaction solution 1: Tris-HCl (pH 8.9) 10mmol / L, KCl 50mmol / L, MgCl 2 4mmol / L, dNTPs each 150umol / L, BSA 0.2μg / μl, Primer1-F 100nmol / L, Primer1-R100nmol / L, Probe175nmol / L, Primer2-F 100nmol / L, Primer2-R 100nmol / L, Probe2 75nmol / L L, Primer3-F100nmol / L, Primer3-R100nmol / L, Probe375nmol / L, Primer7-F75nmol / L, Primer7-R75nmol / L, Probe750nmol / L.

[0058] Enzyme mixture: Taq DNA polymerase 3.81U / μl, UNG 0.224U / μl, anti-Taq DNA polymerase antibody 0.63U / μl.

[0059] After thoroughly mixing 19.5 μl of PCR reaction solution 1 and 0.5 μl of enzyme mixture, dispense 20 μl into the first reaction tube and transfer to the sample processing area. Add 20 μl of the nucleic acid solution obtained in Example 1 into the first reaction tube, cap the re...

Embodiment 3

[0080] Embodiment 3: clinical sample detection

[0081] 30 cases of clinical samples were tested according to the methods of Examples 1 and 2, and the test results are shown in Table 7.

[0082] Table 7. Test results of clinical samples

[0083]

[0084] Note: "+" means positive result, "-" means negative or lower than the detection limit of the reagent, " / " means no result judgment.

[0085] In these 30 samples, the detection results of FAM, VIC, ROX and CY5 channels of PCR reaction solution 1 are as follows: Figure 1~4 Shown; The detection results of FAM, VIC, ROX and CY5 channels of PCR reaction solution 2 are as follows respectively Figure 5-8 shown.

[0086] Depend on Figure 1-8 As shown in Table 7, among the 30 samples, there was 1 case of type A, 10 cases of type B, 7 cases of type C, 3 cases of mixed B / C, 1 case of type D, 3 cases of recombinant C / D and HBV negative 5 cases. The detection result of the present invention is consistent with the clinical detec...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a method, oligonucleotide and kit for HBV genotype detection. A fluorescent PCR technology is adopted, not only can determine whether HBV DNA exists in a sample, but also can perform type identification on HBV genotypes A, B, C and D and C / D recombinant types possibly existing in the sample. In addition, HBV genotypes C and D and C / D recombinant types can be accurately distinguished, and targeted selection of therapeutic drugs is facilitated. Besides, non-competitive internal reference detection is added, the detection process of the whole system is monitored, and false negative results can be effectively prevented.

Description

technical field [0001] The invention belongs to the field of biological science and biotechnology, in particular to a method for detecting HBV genotypes, oligonucleotides and kits. Using fluorescent PCR technology, not only can it be determined whether there is HBV and its viral load in a sample, but also Type identification of HBV genotypes A, B, C, D and C / D recombinants that may exist in the samples. Background technique [0002] Hepatitis B virus (HBV), which belongs to Hepadnaviridae, has a genome length of about 3.2kb, which is a partially double-stranded circular DNA. As a specific hepatotropic virus, due to the development of nucleic acid molecular hybridization technology in recent years, HBV DNA can also be detected in cells of extrahepatic organs. HBV DNA consists of a negative strand (long strand) and a positive strand (short strand). Its positive strand has the function of replication, but not the function of transcription and translation. Its negative strand...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11C12R1/93
CPCC12Q1/706C12Q1/6858C12Q2600/166C12Q2600/156C12Q2531/113C12Q2545/101C12Q2563/107C12Q1/686C12Q2600/16C12Q2561/113C12Q1/6806C12Q1/6844
Inventor 张太松陈斐斐戴明雁洪玉凤
Owner LEADWAY HK
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com