CN109609694B - Kit and method for detecting hepatitis B typing and multiple drug-resistant sites based on Illumina sequencing technology - Google Patents
Kit and method for detecting hepatitis B typing and multiple drug-resistant sites based on Illumina sequencing technology Download PDFInfo
- Publication number
- CN109609694B CN109609694B CN201811635320.9A CN201811635320A CN109609694B CN 109609694 B CN109609694 B CN 109609694B CN 201811635320 A CN201811635320 A CN 201811635320A CN 109609694 B CN109609694 B CN 109609694B
- Authority
- CN
- China
- Prior art keywords
- seq
- primer pair
- sequence
- sequencing
- drug
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Communicable Diseases (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting hepatitis B typing and multiple drug-resistant sites based on an Illumina sequencing technology, which comprises the following steps: a: extracting HBV genome DNA; b: amplifying HBV genome DNA to obtain a first amplification product; c: amplifying the first amplification product by using the second primer pair and the third primer pair to obtain a second amplification product and a third amplification product; d: amplifying the second and third amplification products to obtain a sequencing library; e: performing quality detection quantification on the sequencing library; f: sequencing on a computer; g: and (5) analyzing data to obtain drug-resistant sites and typing related information. The invention also discloses a kit for detecting hepatitis B typing and multiple drug-resistant sites based on the Illumina sequencing technology. The method has the advantages of high sensitivity, strong pertinence, low cost, large flux, high accuracy and the like, can effectively detect the mutation states of a plurality of drug-resistant sites of the HBV sample, and particularly has obvious advantages on low-titer samples.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit and a method for detecting hepatitis B typing and multiple drug resistant sites based on an Illumina sequencing technology.
Background
Hepatitis B Virus (HBV) infection is prevalent worldwide, and about 65 million people die each year from disease caused by HBV infection. In the patients with cirrhosis and stem cell carcinoma in China, the proportion caused by HBV infection is 60% and 80%, respectively. At present, nucleoside (acid) analogues (NA) such as telbivudine (LdT), Adefovir Dipivoxil (ADV), Entecavir (ETV), Tenofovir Disoproxil (TDF) and the like have become one of the main methods for resisting HBV infection. With the long-term application of NAs, virus-resistant strains often appear, which causes difficulty in continuous action of NAs. Therefore, detection in the early stage of drug resistance can often achieve better effect, while the existing detection means often cannot detect low-titer and low-proportion HBV drug resistance mutation, and the technology has certain limitation.
Disclosure of Invention
The invention mainly aims to provide a method for detecting hepatitis B typing and multiple drug-resistant sites based on an Illumina sequencing technology, which can effectively detect the low-titer mutation condition of multiple sites of a sample, and has the advantages of high sensitivity, strong pertinence, low cost, large flux, high accuracy and the like, so as to solve the problem that the existing multiple drug-resistant site mutation detection of HBV virus is difficult to detect the low-titer and low-titer HBV drug-resistant mutation. The second purpose of the invention is to provide a kit for detecting hepatitis B typing and multiple drug-resistant sites based on the Illumina sequencing technology and application thereof. The third purpose of the invention is to provide a system for HBV typing and multi-drug resistant site detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
as a first aspect of the invention, a method for detecting hepatitis B typing and multiple drug resistant sites based on Illumina sequencing technology comprises the following steps:
a: extracting HBV genome DNA in a detection sample;
b: amplifying the HBV genomic DNA using a first primer pair to obtain a first amplification product;
c: amplifying the first amplification product by using a second primer pair and a third primer pair to obtain a second amplification product and a third amplification product;
d: amplifying the second and third amplification products by using an Illumina universal primer pair to obtain a sequencing library;
e: performing quality inspection quantification on the sequencing library;
f: performing sequencing on the machine to obtain a sequencing result;
g: the data analysis obtains the drug-resistant locus and the typing related information,
wherein, the sequence of the first primer pair is shown as SEQ ID NO: 1 and SEQ ID NO: 2, the sequence of the second primer pair is shown as SEQ ID NO: 3 and SEQ ID NO: 4, the sequence of the third primer pair is shown as SEQ ID NO:5 and SEQ ID NO: 6, as shown in SEQ ID NO: 3 and as shown in SEQ ID NO:5, each primer comprises a first tag sequence having a sequence selected from the group consisting of SEQ ID NOs: 9-32, as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 6, each comprising a second tag sequence having a sequence selected from the group consisting of SEQ ID NOs: 33-56. Therefore, the method can effectively detect the mutation states of a plurality of drug-resistant sites of the HBV sample, and has obvious advantages particularly on low-titer samples.
According to the invention, the test sample is a serum or plasma sample of a host sample.
According to the invention, the method further comprises a step of separating and purifying the sequencing library obtained in the step D, wherein the method for separating and purifying is selected from at least one of agarose gel purification, magnetic bead purification, purification column purification and the like.
According to the invention, the sequencer of step F is selected from at least one of the Illumina sequencers, including but not limited to: miseq, Hiseq, Nextseq, Miniseq, Novaseq.
According to the invention, the hepatitis B typing and multi-drug resistance sites are one or more of V173L, L180M, M204V/I, A181T/V, N236T, S202G, M250V, T184G, I169T and A194T.
As a second aspect of the present invention, a kit for detecting hepatitis b typing and multiple drug resistant sites based on Illumina sequencing technology, comprising the nucleic acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, as shown in SEQ ID NO: 3 and SEQ ID NO: 4, and a second primer pair shown as SEQ ID NO:5 and SEQ ID NO: 6.
Further, as shown in SEQ ID NO: 3 and as shown in SEQ ID NO:5, comprising a first tag sequence having a sequence selected from the group consisting of SEQ ID NOs: 9-32, as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 6, each comprising a second tag sequence having a sequence selected from the group consisting of SEQ ID NOs: 33-56.
According to the invention, the multi-drug resistant sites are one or more of V173L, L180M, M204V/I, A181T/V, N236T, S202G, M250V, T184G, I169T and A194T.
As a third aspect of the invention, the application of the kit is to detect the mutation state of the drug-resistant site of the hepatitis B virus.
As a fourth aspect of the present invention, a system for detecting a mutation status of a drug-resistant site of hepatitis b virus, comprising:
an amplification device for use with a nucleic acid sequence as set forth in SEQ ID NO: 1 and SEQ ID NO: 2 to obtain a first amplification product; and then used to utilize the amino acid sequence as set forth in SEQ ID NO: 3 and SEQ ID NO: 4, and a second primer pair shown as SEQ ID NO:5 and SEQ ID NO: 6 to obtain a second amplification product and a third amplification product; and then used again to utilize the amino acid sequence as set forth in SEQ ID NO:57 and SEQ ID NO: amplifying the second and third amplification products by using the Illumina universal primer pair shown in 58 to obtain a sequencing library;
and the sequencing device is connected with the amplification device and is suitable for sequencing the amplification product so as to obtain a sequencing result and determine whether the mutation exists in the HBV nucleic acid sample.
And the analysis device is connected with the sequencing device and is used for determining whether the mutation exists in the HBV nucleic acid sample based on the sequencing result.
Further, the analysis device comprises an alignment unit, in which a reference sequence is stored, and the alignment unit is used for comparing the sequencing result with the reference sequence to determine the mutation of each mutation site of the detection sample.
Furthermore, the system for detecting the mutation state of the hepatitis B virus drug-resistant site also comprises a nucleic acid extraction device, and the nucleic acid extraction device is suitable for separating a hepatitis B virus nucleic acid sample from a hepatitis B virus host.
As a fifth aspect of the present invention, a specific primer pair for detecting a mutation status of a drug-resistant site of hepatitis b virus, comprises the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, as shown in SEQ ID NO: 3 and SEQ ID NO: 4, and a second primer pair shown as SEQ ID NO:5 and SEQ ID NO: 6.
Further, as shown in SEQ ID NO: 3 and as shown in SEQ ID NO:5, comprising a first tag sequence having a sequence selected from the group consisting of SEQ ID NOs: 9-32, as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 6, each comprising a second tag sequence having a sequence selected from the group consisting of SEQ ID NOs: 33-56.
The invention has the beneficial effects that: the method has the advantages of high sensitivity, strong pertinence, low cost, large flux, high accuracy and the like; can effectively detect the mutation states of a plurality of drug-resistant sites of the HBV sample; and the target fragment is obtained by a two-round PCR method, and then the universal primer is used for amplification to form a library, so that the amplification effect is more stable, and the method has obvious advantages particularly on low-titer samples.
Drawings
FIG. 1 is a schematic flow chart of a method for determining the type of mutation site in an HBV nucleic acid sample.
FIG. 2 is a schematic diagram showing the structure of a system for determining the type of mutation site in an HBV nucleic acid sample.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Both the 2 Xgold Star Master Mix and DNase-Free water in the examples of the present invention are commercially available.
1. The design of the primer pairs of the present invention is shown in Table 1
TABLE 1 sequences of primer pairs
| Upstream primer sequence (5 '-3') | SEQ ID | Downstream primer sequence (5 '-3') | SEQ ID | |
| First primer pair | TAGGACCCCTGCTCGTGTTA | NO：1 | GCTAGGAGTTCCGCAGTATGG | NO：2 |
| Second primer set | GACTCGTGGTGGACTTCTCTCA | NO：3 | GTTGTACAGACTTGGCCCCC | NO：4 |
| Third primer set | GGGCTTTCCCCCACTGTY | NO：5 | GRGCAACGGGGTAAAGGK | NO：6 |
| Fourth primer set | TAGGACCCCTGCTCGTGTTA | NO：7 | GGGTTGCGTCAGCAAACAC | NO：8 |
The first primer pair is designed aiming at a plurality of drug-resistant sites of the HBV genome, so that low-titer sample detection sites can be effectively enriched, and amplification products can be amplified by using the second primer pair and the third primer pair.
The second primer pair is designed aiming at a plurality of drug-resistant sites of HBV genome, and the primer comprises a part of Illumina joint sequence so as to adapt to Illumina sequencer.
The third primer pair is designed aiming at a plurality of drug-resistant sites of HBV genome, and the primer comprises a part of Illumina joint sequence so as to adapt to Illumina sequencer.
The fourth primer pair is designed aiming at a plurality of drug-resistant sites of the HBV genome, so that low-titer sample detection sites can be effectively enriched, and amplification products can be amplified by using the second primer pair and the third primer pair.
2. Design of the label of the present invention, see Table 2
TABLE 2 tag sequences
3. Design of Illumina universal primer pair
The universal library PCR primers of Illumina were used, including a universal upstream primer and a downstream primer with a tag (Index) sequence. The sequences of Illumina universal primer pairs are shown in table 3.
PCR was performed using a high efficiency PCR amplification enzyme, with 6 rounds of PCR cycle number selection. After PCR using primers with tag (Index) sequences, libraries from different sources can be pooled and then sequenced on the machine.
TABLE 3 sequences of Illumina Universal primer pairs
Wherein the underlined bases can be combined using a variety of bases according to the Illumina official specifications, thereby creating more, differently tagged primers for the differentiation of different libraries.
The term "high throughput sequencing technology" as used herein refers to second generation sequencing technologies and higher throughput sequencing methods developed thereafter. Second generation sequencing platforms include, but are not limited to, Illumina-Solexa (Miseq, Hiseq, Nextseq, Miniseq, Novaseq, etc.) sequencing platforms, and the like. With the development of sequencing technology, those skilled in the art can understand that other methods and devices for sequencing can also be used for the detection. According to a specific example of the present invention, the nucleic acid tag according to the embodiment of the present invention may be used for sequencing by at least one of Illumina-Solexa sequencing platforms and the like.
The term "nucleic acid" as used in the present invention may be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA. Nucleic acid tags of embodiments of the invention (see table 2). By linking nucleic acid tags to DNA, the source of the DNA sample can be accurately characterized. Tag sequences of more than 3 bases are added to the 5' ends of the upstream and downstream PCR primers respectively, and the upstream and downstream primers containing different tags are combined respectively to distinguish different samples. The label is a base sequence designed artificially, and generally comprises 3 bases or more, and is intended to have a unique sequence label at both ends of a PCR product of each nucleic acid sample, thereby effectively distinguishing PCR products identical in different samples. Thus, by using the nucleic acid tags, DNA libraries of a plurality of samples can be constructed at the same time, the advantage of the sequencing depth can be fully exerted, and mutation states of a plurality of samples can be detected at the same time. For example, the Illumina sequencing technology is utilized, the high throughput of sequencing various DNAs is realized, the efficiency and the throughput of DNA sequencing are improved, and the DNA sequencing cost is reduced.
The invention provides a method for detecting hepatitis B typing and multiple drug-resistant sites based on an Illumina sequencing technology, which comprises the following steps:
step 1: extracting HBV genome DNA in a detection sample by using an extraction kit;
step 2: using the peptide as set forth in SEQ ID NO: 1 and SEQ ID NO: 2 to amplify the HBV genomic DNA to obtain a first amplification product;
and step 3: using the peptide as set forth in SEQ ID NO: 3 and SEQ ID NO: 4 and a second primer pair shown as SEQ ID NO:5 and SEQ ID NO: 6, amplifying the first amplification product to obtain a second amplification product and a third amplification product;
and 4, step 4: using the peptide as set forth in SEQ ID NO:57 and SEQ ID NO: amplifying the second and third amplification products by using the Illumina universal primer pair shown in 58 to obtain a sequencing library;
and 5: performing quality inspection quantification on the sequencing library;
step 6: performing sequencing on the machine to obtain a sequencing result;
and 7: and (5) analyzing data to obtain drug-resistant sites and typing related information.
Wherein, as shown in SEQ ID NO: 3 and the primer shown as SEQ ID NO:5, the primers comprise a first tag sequence which is shown as SEQ ID NO: 9-SEQ ID NO: 32 is shown; as shown in SEQ ID NO: 4 and the primer shown in SEQ ID NO: 6, and the second label sequence is shown as SEQ ID NO: 33-SEQ ID NO: as shown at 56.
In the embodiments of the present invention, the method of introducing the nucleic acid tag into the sequencing library is not particularly limited. The tag may be introduced into the sequencing library by conventional methods during the library construction process. The method can also be used for preprocessing a nucleic acid sample to be sequenced, introducing a tag into an amplification product by a PCR amplification method, and then constructing a sequencing library aiming at the obtained amplification product containing the tag by a conventional library construction method so as to obtain the sequencing library containing the tag. Thus, after a plurality of nucleic acid samples are amplified, they can be mixed together to construct a library, and a sequencing library of a plurality of nucleic acid samples containing respective nucleic acid tag sequences can be easily obtained.
In the embodiment of the present invention, the source of the sample is not particularly limited. The HBV nucleic acid sample is isolated from a host of HBV, preferably, the HBV nucleic acid sample is isolated from a serum or plasma sample of a hepatitis B patient.
In the embodiment of the present invention, the extracted HBV genomic DNA is used as a template for amplification, wherein the polymerase used includes, but is not limited to, conventional taq enzyme, pfu high fidelity enzyme or other polymerase, preferably pfu high fidelity enzyme. And carrying out agarose gel electrophoresis on the amplification product to confirm the amplification condition.
In the embodiment of the present invention, the amplification product of step 4 is further purified and recovered by methods including, but not limited to, agarose gel recovery, magnetic bead purification and purification column purification.
In the embodiment of the invention, in order to effectively improve the purity of the product in each step of library construction, reduce impurity interference and facilitate the subsequent steps, the product in each step of library preparation can be purified and recovered, and the purification method includes but is not limited to magnetic bead purification, purification column purification, agarose gel electrophoresis purification, and preferably magnetic bead purification.
In the embodiment of the present invention, the purified and recovered product is quantified by using a fluorescence quantitative analyzer, including but not limited to, Qubit, and then the products of different samples are mixed according to the same molar number to obtain a mixed amplification product.
In the examples of the present invention, Q-PCR was used for quality control and quantification of the sequencing library, wherein IluminaP5, P7 were used as primers and Illumina phix control kit v3 was used as a standard.
In the embodiment of the invention, sequencing is carried out by a second-generation sequencing platform, preferably an Illumina Miseq platform, and data analysis is carried out to determine whether mutation exists.
In the embodiment of the invention, the universal library-building PCR primer of Illumina company is used, and comprises a universal upstream primer with a sequence shown in SEQ ID NO:57, and a downstream primer with a tag (Index) sequence as shown in SEQ ID NO:58, PCR was performed using a high efficiency PCR amplification enzyme, with 6 rounds of PCR cycle selection. After PCR using primers with tag (Index) sequences, libraries from different sources can be pooled and then sequenced on the machine.
The method can effectively detect the mutation states of a plurality of drug-resistant sites of the HBV sample, and particularly has obvious advantages on low-titer samples. The plurality of drug resistant sites capable of detecting the RT region of the hepatitis B virus genome are at least one selected from V173L, L180M, M204V/I, A181T/V, N236T, S202G, M250V, T184G, I169T and A194T.
The invention also provides a kit for HBV typing and multi-drug resistant site detection, which comprises: as shown in SEQ ID NO: 1 and SEQ ID NO: 2, as shown in SEQ ID NO: 3 and SEQ ID NO: 4, and a second primer pair shown as SEQ ID NO:5 and SEQ ID NO: 6.
The invention also provides a system for HBV typing and multi-drug resistant site detection, which comprises: an amplification device, a sequencing device, and an analysis device. Wherein:
the amplification device is used for amplifying the nucleic acid sample so as to obtain a first amplification product, further obtain a second amplification product and a third amplification product, and further obtain a sequencing library;
a sequencing device is connected with the amplification device and is suitable for sequencing the amplification product so as to obtain a sequencing result;
and the analysis device is connected with the sequencing device and is used for determining whether the mutation exists in the HBV nucleic acid sample based on the sequencing result.
Example 1
In this embodiment, the method for detecting HBV sample in patient serum by using Illumina sequencing technology comprises the following steps:
step 1, according to the instructions, extracting HBV genomic DNA from serum samples of hepatitis B patients by using a TIANAmp virus genomic DNA extraction kit (DP 315, Limited public company of Tiangen Biochemical technology, Beijing).
Step 2, using the nucleotide sequence shown as SEQ ID NO: 1 and SEQ ID NO: 2, amplifying the sample by the first primer pair shown in the figure. The PCR amplification system in step 2 is shown in Table 4.
TABLE 4 PCR amplification System of step 2
| Composition (I) | Volume (μ l) |
| DNA | 5 |
| 2×Gold Star Master Mix | 10 |
| Forward primer F (1. mu.M) | 1 |
| Reverse primer R (1. mu.M) | 1 |
| DNase-Free water | 3 |
The reaction conditions of the PCR of the step 2 are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and total amplification for 35 cycles; final extension at 72 ℃ for 5 min. Thereby, a first amplification product is obtained.
And 3, respectively using the nucleotide sequences shown as SEQ ID NO: 3 and SEQ ID NO: 4 and a second primer pair shown as SEQ ID NO:5 and SEQ ID NO: 6 and a third pair of primers with different tag sequences for different samples.
In this example, the multiple drug resistance related mutation sites contained in the two fragments are detected for 10 samples, and the samples are distinguished by using tag sequences, so that the third primer and the fifth primer have 10 tag sequences, and the 10 tag sequences are shown in SEQ ID NO: 9-SEQ ID NO: 18, the fourth primer and the sixth primer have 1 tag sequence, and the 1 tag sequence is shown as SEQ ID NO: 33, each sample has a unique combination of tag sequences. The amplification system in step 3 is shown in Table 5.
TABLE 5 PCR amplification System of step 3
| Composition (I) | Volume (μ l) |
| DNA | 5 |
| 2×Gold Star Master Mix | 10 |
| Forward primer F (1. mu.M) | 1 |
| Reverse primer R (1. mu.M) | 1 |
| DNase-Free water | 3 |
The reaction conditions of the PCR of the step 3 are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and amplification for 18 cycles; final extension at 72 ℃ for 5 min. Thus, a PCR product was obtained.
And 4, adopting 1.5% agarose gel electrophoresis to confirm the condition of the PCR amplification product in the step 3, wherein 5 mu l of the PCR amplification product in the step 3 is used, the voltage is 120V, the electrophoresis is carried out for 30min, and a gel imaging system is adopted for photographing and detection.
Step 5, the PCR product is recovered by AxyPrepTMThe PCR Clean-up Kit (Axygen) Kit was recovered. The procedure is shown in table 6 below:
TABLE 6 recovery of PCR products
Step 6, taking 1 μ l of the recovered product of step 5, and using
2.0(Invitrogen), and then the products of the different samples were mixed in the same molar ratio to obtain mixed PCR products.
Step 7, taking 20ng of the mixed PCR product, carrying out PCR, and amplifying by using a universal primer of Illumina company, wherein the sequence of the universal primer of Illumina company is shown as SEQ ID NO:57 and SEQ ID NO:58, and the cycle number is 6 rounds, so as to obtain a sequencing library;
the DNA obtained in the previous step was used to prepare a PCR reaction system in a 0.2ml centrifuge tube according to Table 7:
TABLE 7 reaction System for PCR of step 7
The reaction conditions for the PCR of step 7 are shown in Table 8.
TABLE 8 reaction conditions for PCR in step 7
And 8, after the reaction is finished, purifying by using Agencour AMPure XP beads, wherein the specific steps are shown in Table 9.
TABLE 9 purification procedure
And 9, detecting and quantifying library quality.
The library obtained in the last step
2.0(Invitrogen) and Q-PCR for quality control.
And step 10, performing computer sequencing and data analysis.
The samples were subjected to double-ended sequencing using Illumina Miseq PE-300 program to obtain sequencing results, the detailed operational flow is described in Miseq instructions.
And 11, analyzing data.
The sequencing result produced by Miseq is a DNA sequence in a fastq form, the sequencing sequence corresponds to each sample through a sequencing library tag (Index) and a sample tag sequence (barcode), then the obtained DNA sequence is compared with a reference sequence of HBV, finally, the mutation condition of each mutation site of the fragment to be tested is analyzed, and the specific result is shown in Table 10.
Table 10 analysis results of mutation status of each mutation site of the fragments to be tested
| Sample(s) | 169T | 173L | 180M | 181T | 181V | 184G | 194T | 202G | 204I | 204V | 236T | 250V |
| R0020 | 0.02 | 0.16 | 0.12 | 0.07 | 0.03 | 0.00 | 0.04 | 0.10 | 1.88 | 0.05 | 1.37 | 0.05 |
| R0021 | 0.03 | 0.81 | 87.20 | 12.19 | 0.12 | 0.00 | 0.37 | 0.08 | 59.68 | 39.57 | 1.10 | 0.03 |
| R0022 | 0.16 | 0.20 | 98.84 | 0.51 | 0.04 | 0.02 | 0.28 | 0.11 | 0.05 | 99.48 | 0.76 | 0.03 |
| R0023 | 0.02 | 0.45 | 98.55 | 0.28 | 0.02 | 0.00 | 0.13 | 1.45 | 0.03 | 99.47 | 0.54 | 0.07 |
| R0024 | 0.03 | 0.19 | 0.22 | 0.10 | 0.04 | 0.00 | 0.09 | 0.15 | 0.19 | 0.10 | 0.74 | 0.08 |
| R0025 | 0.13 | 0.12 | 98.9 | 44.28 | 0.59 | 0.00 | 0.07 | 0.30 | 0.01 | 99.40 | 0.15 | 0.00 |
| R0026 | 99.21 | 0.32 | 98.14 | 0.30 | 0.04 | 0.00 | 0.12 | 0.10 | 0.35 | 0.00 | 2.13 | 0.05 |
| R0027 | 0.04 | 0.63 | 86.81 | 0.06 | 0.09 | 0.00 | 0.04 | 0.07 | 98.81 | 0.03 | 4.75 | 0.18 |
| R0028 | 0.02 | 0.32 | 19.11 | 0.17 | 0.10 | 0.00 | 0.03 | 0.09 | 99.35 | 0.08 | 0.54 | 0.03 |
| R0029 | 0.01 | 0.19 | 0.19 | 1.40 | 0.00 | 0.00 | 0.47 | 0.03 | 0.63 | 0.09 | 0.85 | 0.01 |
And (4) conclusion: the method can effectively detect the mutation states of a plurality of drug-resistant sites of the HBV samples, and can detect one or more of I169T, V173L, L180M, A181T/V, T184G, A194T, S202G and M204V/I, AN236T as a plurality of drug-resistant sites of the RT region of the hepatitis B virus genome.
Example 2
In this example, two amplification schemes are used to detect a standard substance diluted in a gradient, the standard substance is selected from site-directed mutagenesis artificial plasmids containing a sequence to be detected, and the specific operation steps are as follows:
the first scheme is as follows:
1. to 108The IU/ml standard substance is diluted to 10 degrees in a gradient way7IU/ml、106IU/ml、105IU/ml、104IU/ml、103IU/ml、102IU/ml、10IU/ml。
2. The test results of the standards after the gradient dilution were obtained by the procedure of example 1 are shown in table 11.
Table 11 analysis results of mutation status of each mutation site of the fragments to be tested
The results show that: the concentration of 10 can be detected by adopting the operation step of the first scheme2IU/ml of sample.
And (4) conclusion: the method can effectively detect HBV nucleic acid samples of hepatitis B patients, and the titer of the detectable HBV nucleic acid samples can be as low as 102IU/ml。
Scheme II:
1. to 108IU/ml standard substance, wherein the standard substance is selected from site-directed mutagenesis artificial plasmids containing the sequence to be detected, and the standard substance is subjected to gradient dilution to 107IU/ml、106IU/ml、105IU/ml、104IU/ml、103IU/ml、102IU/ml、10IU/ml。
2. Using the sequences as set forth in SEQ ID NOs: 3 and SEQ ID NO: 4 and a second primer pair shown as SEQ ID NO:5 and SEQ ID NO: 6, and using primers with different tag sequences for different samples.
In this example, the multiple drug resistance related mutation sites contained in the two fragments are detected for 10 samples, and the samples are distinguished by using tag sequences, so that the third primer and the fifth primer have 10 tag sequences, and the 10 tag sequences are shown in SEQ ID NO: 9-SEQ ID NO: 18, the fourth primer and the sixth primer have 1 tag sequence, and the 1 tag sequence is shown as SEQ ID NO: 33, each sample has a unique combination of tag sequences.
The PCR amplification system for this step is shown in Table 12.
TABLE 12 PCR amplification System
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 1min, and amplification for 18 cycles; final extension at 72 ℃ for 5 min. Thus, a PCR product was obtained.
The subsequent steps (e.g., confirmation of PCR amplification product, recovery of PCR product, obtaining of sequencing library, purification, quality inspection and quantification of library, data analysis, etc.) are the same as those in example 1. The data analysis results are shown in table 13.
Table 13 analysis results of mutation status of each mutation site of the fragments to be tested
And (4) conclusion: the concentration can be detected by adopting the operation steps of the second scheme105IU/ml of sample.
Example 3
In this example, two schemes are used to detect a standard substance diluted in a gradient, the standard substance is selected from site-directed mutagenesis artificial plasmids containing a sequence to be detected, and the specific operation steps are as follows:
the first scheme is as follows:
1. to 108The IU/ml standard substance is diluted to 10 degrees in a gradient way7IU/ml、106IU/ml、105IU/ml、104IU/ml、103IU/ml、102IU/ml、10IU/ml。
2. And (3) detecting the standard product after the gradient dilution by using the operation steps of example 1, wherein during the PCR amplification in step 3, a plurality of drug resistance related mutation sites contained in the two fragments are detected for 8 samples, and the samples are distinguished by using tag sequences, so that the third primer and the fifth primer have 8 tag sequences, and the 8 tag sequences are shown as SEQ ID NO: 9-SEQ ID NO: 16, the fourth primer and the sixth primer have 1 tag sequence, and the 1 tag sequence is shown as SEQ ID NO: 33, each sample had a unique combination of tag sequences, and the results are shown in table 14.
Table 14 analysis results of mutation status of each mutation site of the fragments examined
And (4) conclusion: the concentration of 10 can be detected by adopting the operation step of the first scheme2IU/ml of sample.
Scheme II:
1. to 108IU/ml standard substance, wherein the standard substance is selected from site-directed mutagenesis artificial plasmids containing the sequence to be detected, and the standard substance is subjected to gradient dilution to 107IU/ml、106IU/ml、105IU/ml、104IU/ml、103IU/ml、102IU/ml、10IU/ml。
2. The assay was performed on the above-described serially diluted standards using the procedure of example 1, wherein the standard set forth in SEQ ID NO: 7 and SEQ ID NO: the fourth primer set shown in FIG. 8 was used in place of the first primer set in step 2 of example 1 to amplify the standard. The PCR amplification system is shown in Table 15.
TABLE 15 PCR amplification System
The reaction conditions of PCR were: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min, and total amplification for 35 cycles; final extension at 72 ℃ for 5 min. Thereby, a first amplification product is obtained.
The subsequent steps (e.g., PCR amplification product confirmation, PCR product recovery, sequencing library acquisition, purification, library quality determination, data analysis, etc. in step 3) are the same as those in example 1. The data analysis results are shown in table 16.
Table 16 analysis results of mutation status of each mutation site of the fragments to be tested
And (4) conclusion: the concentration of 10 can be detected by adopting the operation steps of the second scheme4IU/ml of sample.
In conclusion, the detection method of the embodiment 1 can detect the mutation condition of a plurality of sites of a low-titer sample at a low proportion, and has the advantages of high sensitivity, strong pertinence, low cost, high flux, high accuracy and the like. The specific primer pair and the label can be further prepared into a detection kit by a method known in the field, which is obvious to those skilled in the art.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. A method for detecting hepatitis B typing and multiple drug-resistant sites based on Illumina sequencing technology for non-diagnosis purposes is characterized by comprising the following steps:
a: extracting HBV genome DNA in a detection sample;
b: amplifying the HBV genomic DNA using a first primer pair to obtain a first amplification product;
c: amplifying the first amplification product by using a second primer pair and a third primer pair to obtain a second amplification product and a third amplification product;
d: amplifying the second and third amplification products by using an Illumina universal primer pair to obtain a sequencing library;
e: performing quality inspection quantification on the sequencing library;
f: performing sequencing on the machine to obtain a sequencing result;
g: the data analysis obtains the drug-resistant locus and the typing related information,
wherein, the sequence of the first primer pair is shown as SEQ ID NO: 1 and SEQ ID NO: 2, the sequence of the second primer pair is shown as SEQ ID NO: 3 and SEQ ID NO: 4, the sequence of the third primer pair is shown as SEQ ID NO:5 and SEQ ID NO: 6 is shown in the specification;
wherein the nucleotide sequences of the Illumina universal primer pair are respectively shown as SEQ ID NO:57 and as shown in SEQ ID NO: shown at 58;
as shown in SEQ ID NO: 3 and as shown in SEQ ID NO:5, each of the primers comprises a first tag sequence at the 5' end, the first tag sequence having a sequence selected from the group consisting of SEQ ID NO: 9-32, as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 6, the 5' ends of the primers each comprise a second tag sequence having a sequence selected from the group consisting of SEQ ID NO: 33-56.
2. The method of claim 1, further comprising a step of performing separation and purification on the sequencing library of step D, wherein the separation and purification is performed by at least one method selected from the group consisting of agarose gel purification, magnetic bead purification, and purification column purification.
3. A kit for detecting hepatitis B typing and multiple drug-resistant sites based on an Illumina sequencing technology is characterized by comprising a first primer pair, a second primer pair and a third primer pair, wherein the sequences of the first primer pair are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, and the sequences of the second primer pair are respectively shown as SEQ ID NO: 3 and SEQ ID NO: 4, and the sequences of the third primer pair are respectively shown as SEQ ID NO:5 and SEQ ID NO: 6 is shown in the specification;
as shown in SEQ ID NO: 3 and as shown in SEQ ID NO:5 comprises a first tag sequence having a sequence selected from the group consisting of SEQ ID NO: 9-32, as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 6, the 5' ends of the primers each comprise a second tag sequence having a sequence selected from the group consisting of SEQ ID NO: 33-56.
4. Use of the kit of claim 3 for the preparation of a reagent for detecting the mutational status of the drug resistant site of hepatitis b virus.
5. A specific primer pair combination for detecting the mutation state of a hepatitis B virus drug-resistant site is characterized by comprising a primer shown as SEQ ID NO: 1 and SEQ ID NO: 2, as shown in SEQ ID NO: 3 and SEQ ID NO: 4 and a second primer pair shown as SEQ ID NO:5 and SEQ ID NO: 6, a third primer pair; as shown in SEQ ID NO: 3 and as shown in SEQ ID NO:5, each of the primers comprises a first tag sequence at the 5' end, the first tag sequence having a sequence selected from the group consisting of SEQ ID NO: 9-32, as shown in SEQ ID NO: 4 and as shown in SEQ ID NO: 6, the 5' ends of the primers each comprise a second tag sequence having a sequence selected from the group consisting of SEQ ID NO: 33-56.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811635320.9A CN109609694B (en) | 2018-12-29 | 2018-12-29 | Kit and method for detecting hepatitis B typing and multiple drug-resistant sites based on Illumina sequencing technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811635320.9A CN109609694B (en) | 2018-12-29 | 2018-12-29 | Kit and method for detecting hepatitis B typing and multiple drug-resistant sites based on Illumina sequencing technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109609694A CN109609694A (en) | 2019-04-12 |
| CN109609694B true CN109609694B (en) | 2022-03-01 |
Family
ID=66015339
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811635320.9A Active CN109609694B (en) | 2018-12-29 | 2018-12-29 | Kit and method for detecting hepatitis B typing and multiple drug-resistant sites based on Illumina sequencing technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109609694B (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001094559A1 (en) * | 2000-06-09 | 2001-12-13 | Melbourne Health | Hepatitis b virus dna polymerase and surface antigen variants and methods of using same |
| CN1384888A (en) * | 1999-07-08 | 2002-12-11 | 基因创新有限公司 | Detection of anti-hepatitis drug resistance |
| CN101545013A (en) * | 2009-03-10 | 2009-09-30 | 上海翼和应用生物技术有限公司 | Hepatitis B virus multi-drug resistant gene locus typing detection kit |
| CN101812537A (en) * | 2009-10-27 | 2010-08-25 | 重庆医科大学 | Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus |
| CN102181575A (en) * | 2011-03-18 | 2011-09-14 | 中国科学院武汉病毒研究所 | Primers and method for detecting drug resistance mutation site of hepatitis B virus |
| CN102758026A (en) * | 2012-06-29 | 2012-10-31 | 深圳华大基因科技有限公司 | HiSeq sequencing technology-based method for detecting hepatitis B virus type and drug resistance gene |
| CN104878125A (en) * | 2015-06-18 | 2015-09-02 | 上海昂朴生物科技有限公司 | High throughput testing method of multiple drug-resisting sites of hepatitis B virus |
| CN105316403A (en) * | 2015-05-12 | 2016-02-10 | 深圳市瀚海基因生物科技有限公司 | Primer, method and application based on next-generation sequencing technology to detect HBV drug-resistant mutation site |
| CN105603122A (en) * | 2016-01-22 | 2016-05-25 | 上海同科生物科技有限公司 | Hepatitis B virus YMDD gene mutation detection kit |
| US9702005B2 (en) * | 2002-04-12 | 2017-07-11 | Abl Sa | Hepatitis B viral variants with reduced susceptibility to nucleoside analogs and uses thereof |
-
2018
- 2018-12-29 CN CN201811635320.9A patent/CN109609694B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1384888A (en) * | 1999-07-08 | 2002-12-11 | 基因创新有限公司 | Detection of anti-hepatitis drug resistance |
| WO2001094559A1 (en) * | 2000-06-09 | 2001-12-13 | Melbourne Health | Hepatitis b virus dna polymerase and surface antigen variants and methods of using same |
| US9702005B2 (en) * | 2002-04-12 | 2017-07-11 | Abl Sa | Hepatitis B viral variants with reduced susceptibility to nucleoside analogs and uses thereof |
| CN101545013A (en) * | 2009-03-10 | 2009-09-30 | 上海翼和应用生物技术有限公司 | Hepatitis B virus multi-drug resistant gene locus typing detection kit |
| CN101812537A (en) * | 2009-10-27 | 2010-08-25 | 重庆医科大学 | Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus |
| CN102181575A (en) * | 2011-03-18 | 2011-09-14 | 中国科学院武汉病毒研究所 | Primers and method for detecting drug resistance mutation site of hepatitis B virus |
| CN102758026A (en) * | 2012-06-29 | 2012-10-31 | 深圳华大基因科技有限公司 | HiSeq sequencing technology-based method for detecting hepatitis B virus type and drug resistance gene |
| CN105316403A (en) * | 2015-05-12 | 2016-02-10 | 深圳市瀚海基因生物科技有限公司 | Primer, method and application based on next-generation sequencing technology to detect HBV drug-resistant mutation site |
| CN104878125A (en) * | 2015-06-18 | 2015-09-02 | 上海昂朴生物科技有限公司 | High throughput testing method of multiple drug-resisting sites of hepatitis B virus |
| CN105603122A (en) * | 2016-01-22 | 2016-05-25 | 上海同科生物科技有限公司 | Hepatitis B virus YMDD gene mutation detection kit |
Non-Patent Citations (2)
| Title |
|---|
| Advances in Molecular Diagnosis of HBV Infection and Drug Resistance;Erwin Sablon等;《Int. J. Med. Sci.》;20050105;第2卷(第1期);8-16 * |
| MBL-2基因多态性与HBV相关肝病的发生发展有关;顾星等;《第一次全国中西医结合检验医学学术会议暨中国中西医结合学会检验医学专业委员会成立大会论文汇编》;20140612;212-213 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109609694A (en) | 2019-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107177670B (en) | Method for detecting Parkinson disease pathogenic gene mutation in high throughput manner | |
| CN111254190B (en) | Nanopore third-generation sequencing detection method for plasma virology | |
| CN108998508B (en) | Construction method of amplicon sequencing library, primer group and kit | |
| CN104878125B (en) | A kind of high-flux detection method for many resistance sites of hepatitis type B virus | |
| CN106554955B (en) | Method and kit for constructing sequencing library of PKHD1 gene mutation and application thereof | |
| CN111073961A (en) | High-throughput detection method for gene rare mutation | |
| WO2018133546A1 (en) | CONSTRUCTION METHOD, DETECTION METHOD AND KIT FOR NON-INVASIVE PRENATAL FETAL α-THALASSEMIA GENE MUTATION DETECTION LIBRARY | |
| CN116024208B (en) | Kit capable of simultaneously detecting 26 pig epidemic diseases through single reaction | |
| CN111424119B (en) | High-flux detection primer and kit for SARS-CoV-2 virus | |
| CN110724731A (en) | Method for adding internal reference quantity of nucleic acid copy number in multiplex PCR system | |
| CN103998625B (en) | For the method and system of Viral diagnosis | |
| CN106906210A (en) | A kind of fusion primer combination of rapid build amplification sublibrary | |
| CN115948607B (en) | Method and kit for simultaneously detecting multiple pathogen genes | |
| CN111944806A (en) | Molecular tag group for high-throughput sequencing pollution detection and application thereof | |
| CN109609694B (en) | Kit and method for detecting hepatitis B typing and multiple drug-resistant sites based on Illumina sequencing technology | |
| CN105296471B (en) | DNA label, PCR primer and application thereof | |
| CN111304309A (en) | Detection method for sequencing platform tag sequence pollution | |
| CN116463408A (en) | ABO gene amplification primer, amplification system, amplification method, sequencing library construction method and sequencing method | |
| CN107164365B (en) | Method for detecting chromosome telomere DNA full length by applying third generation sequencing technology | |
| EP3853362A1 (en) | System and method for modular and combinatorial nucleic acid sample preparation for sequencing | |
| CN117265091B (en) | Primer group, kit and application for HLA-DRB3/4/5 genotyping | |
| CN114015757B (en) | Method for detecting fusion gene | |
| CN117512085B (en) | Primer group and kit for detecting HLA-DPB1 genotyping | |
| CN117265090B (en) | Primer set and kit for detecting HLA-DQA1 genotyping of human leukocyte antigen | |
| CN114774514B (en) | Library construction method and kit suitable for high-throughput targeted genome methylation detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |