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

Kit and method for detecting hepatitis B typing and multi-drug resistance loci based on illumina sequencing technology

A technique for detection of hepatitis B, which is applied in the field of kits for detection of hepatitis B typing and multi-drug resistance sites, can solve problems such as difficult detection of low-titer HBV drug resistance mutations, and achieve stable amplification effect, The effect of high accuracy and low cost

Active Publication Date: 2022-03-01
上海昂朴生物科技有限公司 +2
View PDF10 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The primary purpose of the present invention is to provide a method for detecting hepatitis B typing and multi-drug resistance sites based on Illumina sequencing technology, which can effectively detect low-ratio mutations at multiple sites in low-titer samples, and has High sensitivity, strong pertinence, low cost, large throughput, high accuracy, etc., to solve the problem that the existing multi-drug resistance site mutation detection of HBV virus is difficult to detect low-titer, low-proportion HBV drug-resistant mutations

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
  • Kit and method for detecting hepatitis B typing and multi-drug resistance loci based on illumina sequencing technology
  • Kit and method for detecting hepatitis B typing and multi-drug resistance loci based on illumina sequencing technology
  • Kit and method for detecting hepatitis B typing and multi-drug resistance loci based on illumina sequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] In this embodiment, Illumina sequencing technology is used to detect HBV samples in patient serum, and the specific operation steps are as follows:

[0080] Step 1. According to the instructions, use the TIANamp Viral Genomic DNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP315) to extract HBV genomic DNA from serum samples of hepatitis B patients.

[0081] Step 2, using the first primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 to amplify the sample. The PCR amplification system of step 2 is shown in Table 4.

[0082] Table 4 PCR amplification system of step 2

[0083] Element Volume (μl) dna 5 2×Gold Star Master Mix 10 Forward primer F (1μM) 1 Reverse primer R (1μM) 1 DNase-Free water 3

[0084] The reaction conditions of PCR in step 2 were: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, a total of 35 cycles of amplif...

Embodiment 2

[0116] In this embodiment, two kinds of amplification schemes are used to detect the standard substance of gradient dilution. The standard substance is selected from the site-directed mutagenesis artificial plasmid containing the sequence to be detected. The specific operation steps are as follows:

[0117] Option One:

[0118] 1, against 10 8 IU / ml standard was serially diluted to 10 7 IU / ml, 10 6 IU / ml, 10 5 IU / ml, 10 4 IU / ml, 10 3 IU / ml, 10 2 IU / ml, 10IU / ml.

[0119] 2. Using the operation steps of Example 1 to detect the standard substance after the gradient dilution, the results are shown in Table 11.

[0120] The analysis result of the mutation situation of each mutation site of the measured fragment in Table 11

[0121]

[0122] The results show that the operation steps of Scheme 1 can detect the concentration of 10 2 IU / ml of sample.

[0123] Conclusion: This method can effectively detect HBV nucleic acid samples from hepatitis B patients, and the detectab...

Embodiment 3

[0138] In this example, two schemes are used to detect the standard substance of gradient dilution. The standard substance is selected from the site-directed mutagenesis artificial plasmid containing the sequence to be detected. The specific operation steps are as follows:

[0139] Option One:

[0140] 1, against 10 8 IU / ml standard was serially diluted to 10 7 IU / ml, 10 6 IU / ml, 10 5 IU / ml, 10 4 IU / ml, 10 3 IU / ml, 10 2 IU / ml, 10IU / ml.

[0141] 2. Using the operation steps of Example 1 to detect the above-mentioned standard substance after gradient dilution, wherein, during the PCR amplification in step 3, multiple drug-resistance-related mutations contained in the above-mentioned two fragments were detected for 8 samples Point, using the tag sequence to distinguish samples, therefore, the third primer and the fifth primer have 8 tag sequences, these 8 tag sequences are shown in SEQ ID NO: 9 - SEQ ID NO: 16, the fourth primer, the sixth primer The primer has a tag sequ...

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 discloses a method for detecting hepatitis B typing and multi-drug resistance sites based on Illumina sequencing technology, comprising the following steps: A: extracting HBV genomic DNA; B: amplifying HBV genomic DNA to obtain the first amplification Amplified products; C: Use the second and third primer pairs to amplify the first amplified product to obtain the second and third amplified products; D: amplify the second and third amplified products to obtain a sequencing library; E: pair Quality inspection and quantification of the sequencing library; F: Sequencing on the machine; G: Data analysis to obtain information about drug resistance sites and genotyping. The invention also discloses a kit for detecting hepatitis B typing and multi-drug resistance sites based on Illumina sequencing technology. The method of the present invention has the advantages of high sensitivity, strong pertinence, low cost, large throughput, and high accuracy, and can effectively detect the mutation status of multiple drug-resistant sites in HBV samples, especially in low-titer samples. .

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit and a method for detecting hepatitis B typing and multi-drug resistance sites based on Illumina sequencing technology. Background technique [0002] Hepatitis B virus (HBV) infection is a worldwide epidemic, and about 650,000 people die from diseases caused by HBV infection every year. HBV infection accounts for 60% and 80% of patients with liver cirrhosis and stem cell carcinoma in China, respectively. At present, nucleoside (acid) analogs (NA) such as telbivudine (LdT), adefovir dipivoxil (ADV), entecavir (ETV), tenofovir dipivoxil (TDF), etc., have become anti-HBV infection one of the main methods. With the long-term application of NAs, drug-resistant strains of the virus often appear, which makes it difficult for NAs to continue to work. Therefore, detection in the early stage of drug resistance can often achieve better results, but the current detection meth...

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 Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/706C12Q2531/113C12Q2535/122C12Q2537/165
Inventor 窦同海高春芳王曦路祝海军陈雯雯陈静高惠芳
Owner 上海昂朴生物科技有限公司
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