Illumina sequencing technology based kit and method for detecting hepatitis B typing and multi-drug resistant sites

A technology for hepatitis B and technical detection, applied in the biological field, can solve problems such as the difficulty in detecting low-titer HBV drug-resistant mutations, and achieve stable amplification effects, strong pertinence, and high sensitivity

Active Publication Date: 2019-04-12
上海昂朴生物科技有限公司 +2
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  • 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

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  • Illumina sequencing technology based kit and method for detecting hepatitis B typing and multi-drug resistant sites
  • Illumina sequencing technology based kit and method for detecting hepatitis B typing and multi-drug resistant sites
  • Illumina sequencing technology based kit and method for detecting hepatitis B typing and multi-drug resistant sites

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

[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 amplification; and final extension at 72°C for 5 min. Thus, a first amplification product is obtained.

[0085] Step 3, respectively using the second prim...

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...

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Abstract

The invention discloses an Illumina sequencing technology based method for detecting hepatitis B typing and multi-drug resistant sites. The method comprises the following steps: A: extracting HBV genomic DNA; B: amplifying HBV genomic DNA to obtain a first amplification product; C: amplifying the first amplification product using second and third primer pairs to obtain second and third amplification products; D: amplifying the second and third amplification products to obtain a sequencing library; E: subjecting the sequencing library to quality inspection and quantification; F: conducting sequencing on a machine; and G: conducting data analysis to obtain information on drug resistance sites and typing. The invention also discloses an Illumina sequencing technology based kit for detecting the hepatitis B typing and multi-drug resistant sites. The method of the invention has the advantages of high sensitivity, strong pertinence, low cost, large flux, and high accuracy, can effectively detect the mutation state of multiple drug resistance sites of a HBV sample, and especially has obvious advantages on a low titer sample.

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

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Application Information

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Patent Type & Authority Applications(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
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