Hepatitis B virus YMDD motif mutation detection specific primer and liquid phase chip and method thereof

A hepatitis B virus and mutation detection technology, applied in the field of molecular biology, can solve the problems of large-scale, rapid and accurate clinical detection, discovery of hepatitis B virus YMDD mutant strains, and failure to meet the high-throughput requirements of clinical detection, etc. Achieve accurate and reliable detection results, avoid uncertain factors, and improve detection accuracy

Active Publication Date: 2010-01-27
SUREXAM BIO TECH
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Problems solved by technology

Although direct sequencing has always been considered the gold standard for detecting gene mutations, it is time-consuming and laborious, does not meet the high-throughput requirements of clinical testing, and its sensitivity is not high
RFLP also has complicated operation steps, and the test results are affected by many aspects, so it cannot meet the needs of large-scale, rapid and acc

Method used

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  • Hepatitis B virus YMDD motif mutation detection specific primer and liquid phase chip and method thereof
  • Hepatitis B virus YMDD motif mutation detection specific primer and liquid phase chip and method thereof
  • Hepatitis B virus YMDD motif mutation detection specific primer and liquid phase chip and method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 The liquid-phase chip for detecting the mutation of the hepatitis B virus YMDD motif mainly includes:

[0037] 1. Specific primer sequences and ASPE primers

[0038] Specific primer sequences were designed for the six common mutation sites rtL80I / V, rtV173L, rtL180M, rtA181V / T, rtM204V / I / S and rtN236T of the HBV YMDD motif associated with nucleoside analog therapy resistance . ASPE primers consist of Tag+ specific primer sequences. ASPE primer sequences are shown in the following table: (SEQ ID NO.1~SEQ ID NO.33 are specific sequences)

[0039] Table 1 ASPE primer sequence

[0040]

[0041]

[0042] Table 2 The Tag sequence of the 5' end of the ASPE-specific primer

[0043]

[0044]

[0045]

[0046] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere (as shown in Table 2), and the 3' end is a mutant or wild-type specific primer fragment (such as shown in T...

Embodiment 3

[0134] Embodiment 3 Selection of Tag sequence and Anti-Tag sequence:

[0135] 1. Design of liquid phase chip preparation

[0136] Taking the detection liquid chip of rtL180M mutation as an example, the specific primers at the 3' end of the ASPE primer are designed for the wild type and mutant type of rtL180M, and the Tag sequence at the 5' end of the ASPE primer is selected from SEQ ID NO.33-SEQ ID NO . For 3 of 66, correspondingly, the anti-tag sequences coated on the microspheres and complementary to the corresponding tag sequences are selected from SEQ ID NO.67-SEQ ID NO.99. The specific design is shown in the following table (Table 5). The synthesis of ASPE primers, Anti-tag sequence coated microspheres, amplification primers, etc. are as described in Example 1.

[0137] Table 7

[0138]

[0139] 2. Sample testing

[0140] Using the liquid phase chip prepared by the above design, serum samples 1-10 were detected according to the detection process and method described ...

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Abstract

The invention discloses a hepatitis B virus YMDD motif mutation detection specific primer and liquid phase chip and a method thereof. The liquid phase chip mainly comprises an ASPE primer, a microsphere coated with an anti-tag sequence and a primer used for amplifying target sequences having mutation sites of rtL801/V, rtV173L, rtL180M, rtA181V/T, rtM204V/I/S and/or rtN 236T. The detection liquid phase chip and the detection method have high detection sensitivity, high signal-to-noise ratio and more accurate and reliable detection results. Moreover, detecting 6 mutation sites can be completed in the same reaction, i.e., the operation is convenient, and many uncertain factors existing in many times of operational processes are avoided, thereby greatly improving the detection accuracy.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to specific primers for detecting mutations of viral YMDD motifs related to drug resistance of hepatitis B nucleoside analogs, a liquid phase chip and a detection method thereof. technical background [0002] Hepatitis B is a globally widespread infectious disease with geographical distribution in many countries. There are more than 2 billion people in the world who have been infected with hepatitis B virus (hepatitis B virus, HBV), of which 350-400 million are chronic carriers, and there are 120 million people in China. Chronic hepatitis B can cause liver cirrhosis, liver failure, liver cancer and other serious liver diseases, and about 1 million people die every year in the world. And China is the hardest-hit area of ​​chronic hepatitis B, and more than 8% of the population are chronic carriers, and about 300,000 people die of li...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 许嘉森杨惠夷何嘉英
Owner SUREXAM BIO TECH
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