METHOD FOR DETECTING HBV GENOTYPE, OLIGONUCLEOTIDE AND KIT
20230167514 · 2023-06-01
Assignee
Inventors
- Taisong ZHANG (Hangzhou, Zhejiang, CN)
- Feifei CHEN (Hangzhou, Zhejiang, CN)
- Mingyan DAI (Hangzhou, Zhejiang, CN)
- Yugeng HONG (Hangzhou, Zhejiang, CN)
Cpc classification
C12Q2600/166
CHEMISTRY; METALLURGY
C12Q1/6806
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides a method, an oligonucleotide and a kit for detecting HBV genotypes, which by use of the fluorescent PCR technology, not only can determine the presence or absence of HBV DNA in a sample, but also can achieve the genotype identification of HBV genotypes A, B, C, D and C/D recombinant type that may be present in the sample.
Claims
1. An oligonucleotide for fluorescent PCR detection of HBV genotypes, comprising: (1) a first pair of primers and a first probe for specific detection of HBV genotype D and C/D recombinant type, the first pair of primers and the first probe being designed for the S gene region of the HBV genotype D; and (2) a second pair of primers and a second probe for specific detection of the HBV C/D recombinant type, the second pair of primers and the second probe being designed for the C gene region of the HBV C/D recombinant type.
2. The oligonucleotide of claim 1, wherein the first pair of primers and the first probe have base sequences of SEQ ID NOs: 13 to 15.
3. The oligonucleotide of claim 1, wherein the second pair of primers and the second probe have base sequences of SEQ ID NOs: 16 to 18.
4. The oligonucleotide of claim 1, further comprising at least one of (3) to (6): (3) primers and a probe for detecting HBV genotype A, which have base sequences of SEQ ID NOs: 1 to 3 respectively; (4) primers and a probe for detecting HBV genotype B, which have base sequences of SEQ ID NOs: 4 to 6 respectively; (5) primers and a probe for detecting HBV genotype C, which have base sequences of SEQ ID NOs: 10 to 12 respectively; and (6) primers and a probe for detecting HBV DNA, which have base sequences of SEQ ID NOs: 7 to 9 respectively.
5. The oligonucleotide of claim 1, further comprising: (7) primers and a probe for detecting an internal reference, which have base sequences of SEQ ID NOs: 19 to 21 respectively.
6. A method for detecting HBV genotypes using fluorescent PCR, comprising: (1) extracting DNA from a sample; (2) performing fluorescent PCR amplification on the DNA in step (1) in the presence of a set of primers and probes; (3) determining whether the sample has the HBV genotype D or C/D recombinant type based on the result in step (2), wherein the set of primers and probes comprises a first pair of primers and a first probe for specific detection of the HBV genotype D and C/D recombinant type, and a second pair of primers and a second probe for specific detection of the HBV C/D recombinant type, the first pair of primers and the first probe being designed for the S gene region of the HBV genotype D, and the second pair of primers and the second probe being designed for the C gene region of the HBV C/D recombinant type.
7. The method of claim 6, wherein the set of primers and probes further comprises a pair of primers and a probe for detecting HBV genotype C, which have base sequences of SEQ ID NOs: 10 to 12 respectively.
8. The method of claim 6, further comprising performing fluorescent PCR amplification on the DNA in step (1) in the presence of another set of primers and probes, wherein the another set of primers and probes comprises at least one of 1) to 3): 1) primers and a probe for detecting HBV genotype A, which have base sequences of SEQ ID NOs: 1 to 3 respectively; 2) primers and a probe for detecting HBV genotype B, which have base sequences of SEQ ID NOs: 4 to 6 respectively; and 3) primers and a probe for detecting HBV DNA, which have base sequences of SEQ ID NOs: 7 to 9 respectively.
9. The method of claim 6, wherein the first pair of primers and the first probe have base sequences of SEQ ID NOs: 13 to 15, and the second pair of primers and the second probe have base sequences of SEQ ID NOs: 16 to 18.
10. A kit for detecting HBV genotypes using fluorescent PCR, comprising a fluorescent PCR reaction solution that comprises an oligonucleotide, wherein the oligonucleotide comprises: (1) a first pair of primers and a first probe for specific detection of HBV genotype D and C/D recombinant type, the first pair of primers and the first probe being designed for the S gene region of the HBV genotype D; and (2) a second pair of primers and a second probe for specific detection of the HBV C/D recombinant type, the second pair of primers and the second probe being designed for the C gene region of the HBV C/D recombinant type.
11. The kit of claim 10, wherein the first pair of primers and the first probe have base sequences of SEQ ID NOs: 13 to 15, and the second pair of primers and the second probe have base sequences of SEQ ID NOs: 16 to 18.
12. The kit of claim 10, wherein the oligonucleotide further comprises at least one of (3) to (6): (3) primers and a probe for detecting HBV genotype A, which have base sequences of SEQ ID NOs: 1 to 3 respectively; (4) primers and a probe for detecting HBV genotype B, which have base sequences of SEQ ID NOs: 4 to 6 respectively; (5) primers and a probe for detecting HBV genotype C, which have base sequences of SEQ ID NOs: 10 to 12 respectively; and (6) primers and a probe for detecting HBV DNA, which have base sequences of SEQ ID NOs: 7 to 9 respectively.
13. The kit of claim 10, wherein the oligonucleotide further comprises: (7) primers and a probe for detecting an internal reference, which have base sequences of SEQ ID NOs: 19 to 21 respectively.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE EMBODIMENTS
[0034] Given that different HBV genotypes are related to patients' clinical performance, prognosis and treatment response to a certain extent, it is necessary to perform HBV genotyping detection on an HBV positive sample. The present invention can detect not only HBV virus genotypes A, B, C and D but also the presence or absence of the C/D recombinant type by using the fluorescent quantitative PCR method, so that genotype C, genotype D and the C/D recombinant type can be distinguished.
[0035] The present invention also adds the detection of HBV DNA; as long as HBV is present in the sample, the presence of HVB DNA can be detected regardless of the genotype, avoiding missed detection; and in addition, when one or more of genotypes A, B, C, D and the C/D recombinant type are present in the clinical sample, not only is the fluorescent PCR detection for this one or more genotypes positive, but also the detection for HBV DNA is positive, which can increase the reliability of the sample detection. In the present invention, HBV detection can be performed in two tubes: the first reaction tube can be used for detecting genotype A, genotype B and HBV DNA, the second reaction tube can be used for detecting genotype C, genotype D and the C/D recombinant type, and of course, the objects to be detected in each reaction tube can be appropriately adjusted according to the actual needs. In addition, a non-competitive internal reference is added to each of the first and second reaction tubes to monitor the detection process of the whole system, so that the occurrence of false negative results can be effectively prevented. The non-competitive internal reference added into the first and second reaction tubes can be either the same or different.
[0036] In the present invention, in order to reduce possible non-specific amplification generated during fluorescent PCR amplification as far as possible, an anti-Taq DNA polymerase antibody can be added to a fluorescent PCR reaction solution, and as this antibody can bind the active sites of Taq DNA polymerase, Taq DNA polymerase cannot play its function; and only in the denaturation phase, the anti-Taq DNA polymerase antibody is inactivated at high temperature and cannot bind to the active sites of Taq DNA polymerase, thereby enabling Taq DNA polymerase to play a catalytic role to synthesize new DNA strands.
[0037] The design of the primers and probes of the present invention and its genotyping idea are as follows: the primers and probes designed in the present invention are aimed at selecting multiple gene regions of multiple HBV genotypes, instead of being limited in a single gene region, which helps avoid cross reaction and perform further genotype identification. Specifically, a pair of primers and a probe for detecting HBV DNA are designed for conserved regions of HBV genotypes A to H, and primers and probes for detecting a HBV genotype are designed for specific fragments in the S gene region of a specific HBV genotype; however, given that the C/D recombinant type has partial sequence overlap with genotypes C and D, specific primers and probes designed according to the S gene region of genotype C can detect only genotype C, but not genotype D and the C/D recombinant type. The specific primers and probes designed according to the S gene region of genotype D can detect not only genotype D but also the C/D recombinant type, thus there is a problem that the C/D recombinant type is misdiagnosed as genotype D. Therefore, in order to accurately distinguish genotype D from the C/D recombinant type, the present invention additionally designs a pair of primers and a probe for the C gene region of the C/D recombinant type to distinguish genotype D from the C/D recombinant type.
[0038] For the sake of facilitating illustration, the primers and probes given in Table 1 are taken as examples for illustration. In this way, in the first reaction tube, fluorescent PCR amplification is performed on a clinical sample in four colors to detect HBV genotype A (VIC channel), HBV genotype B (FAM channel), HBV DNA (ROX channel), and internal reference (Cy5 channel), so as to not only detect the presence or absence of HBV in the sample, but also determine the presence or absence of genotype A or genotype B. In the second reaction tube, fluorescent PCR amplification and HBV genotyping detection are performed on the clinical sample in four colors to detect genotype C (FAM channel), genotype D and C/D recombinant type (VIC channel), C/D recombinant type (ROX channel), and reference gene (Cy5 channel), so as to not only detect the presence or absence of genotype C and genotype D in the sample, but also determine the presence or absence of the C/D recombinant type. In the second reaction tube, the VIC channel and the ROX channel can be used to determine the presence or absence of genotype D and the C/D recombinant type. Specifically, given that genotype C, genotype D and the C/D recombinant type have partial sequence overlap, the primers and probe for detecting genotype C can detect only genotype C but not the C/D recombinant type, but the primers and probe for detecting genotype D (VIC channel) can detect not only genotype D but also the C/D recombinant type. Therefore, an additional pair of primers and a probe are designed in the C gene region of the C/D recombinant type to distinguish genotype D from the C/D recombinant type (ROX channel): when both the VIC channel and the ROX channel are positive, it is determined that the C/D recombinant type is present; when the VIC channel is positive but the ROX channel is negative, it is determined that genotype D is present and the C/D recombinant type is absent; and when both the VIC channel and the ROX channel are negative, it is determined that genotype D and the C/D recombinant type are absent. For the other situation in the second reaction tube, i.e., it is impossible that the VIC channel is a negative and the ROX channel is positive, this is because the primers and probe (VIC channel) for detecting genotype D can detect not only genotype D but also the C/D recombinant type, and when the VIC channel is negative, which means the absence of both genotype D and the C/D recombinant type in the sample, at this point, the ROX channel is also negative and impossible to be positive, so there is no need to make further result determination for the ROX channel
TABLE-US-00001 TABLE 1 Primers and probes Primers Sequence Base sequences and probes number (5′.fwdarw.3′) Notes Primer1-F SEQ ID GATTATCAAGGTATG Detecting NO: 1 TTGCCCG genotype A Primer1-R SEQ ID TGCCTTGAGCAGGAG NO: 2 TCGT Probe1 SEQ ID VIC-ACAACAACAAC NO: 3 CAGTACG-MGB Primer2-F SEQ ID AACACCCGTGTGTCT Detecting NO: 4 TGGC genotype B Primer2-R SEQ ID TAACCAGGACAAATT NO: 5 GGAGGA Probe2 SEQ ID FAM-CCAAATCTCCA NO: 6 GTCACT-MGB Primer3-F SEQ ID CTAGGACCCCTGCTC HBV DNA NO: 7 GTGT Primer3-R SEQ ID GAGAGAAGTCCACCA NO: 8 CGAGTCTA Probe3 SEQ ID ROX-CAGGCGGGGTTTT NO: 9 TCTTGTTGAC-BHQ2 Primer4-F SEQ ID CTCGTGGTGGACTTCTC Detecting NO: 10 TCAAT genotype C Primer4-R SEQ ID GTTGGGGACTGCGAAT NO: 11 TTT Probe4 SEQ ID FAM-CACCCAMGTGTC NO: 12 CTG-MGB Primer5-F SEQ ID CTCGTGGTGGACTTC Genotype NO: 13 TCTCAAT D, C/D recombinant type Primer5-R SEQ ID GTTGGGGACTGCGAAT NO: 14 TTT Probe5 SEQ ID VIC-ACACACGGTAGT NO: 15 MTCC-GB Primer6-F SEQ ID ATTTGGAGCTTCC C/D NO: 16 GTGGAGTTA recombinant type Primer6-R SEQ ID GCAGTATGGTGAG NO: 17 GTGAACAATGT Probe6 SEQ ID ROX-CCTTCTATTC NO: 18 GAGATCTCCT CGACACCG-BHQ2 Primer7-F SEQ ID TGCAAGAAGGATCG Reference NO: 19 TTGAAGCT gene Primer7-R SEQ ID CAACAGTCTGGCCT NO: 20 TTCAGCA Probe7 SEQ ID Cy5-ATGAGGGCAGG NO: 21 CCGCTATGATGGAT-BHQ2
[0039] The following examples further illustrate the present invention. These examples are not intended to limit the protection scope of the present invention, but provides further understanding of the present invention.
Example 1: Extraction of Sample DNA
[0040] On the Promotor® NES-32 nucleic acid purifier from Acon Biotech (Hangzhou) Co., Ltd., clinical sample DNA was extracted using reagents in the nucleic acid (DNA) extraction kit (magnetic bead method) from Acon Biotech (Hangzhou) Co., Ltd. 1) The reagent solutions shown in Table 2 were added to the 96-well plate in advance (this step could be omitted if pre-loaded plate reagents were used).
TABLE-US-00002 TABLE 2 Nucleic acid extraction solution 96-well reagent plate Reagent composition Column 1 Column 7 Lysis solution 400 μl Column 2 Column 8 Washing solution 1 600 μl Column 3 Column 9 Washing solution 2 600 μl Column 4 Column 10 Washing solution 2 600 μl Column 5 Column 11 Magnetic bead solution 100 μl Column 6 Column 12 Adding 75 μl elution solution at first, and then adding 30 μl of paraffin oil
[0041] 2) 20 μl of proteinase K was added to column 1 and column 7 of the 96-well plate in order at first; and then 200 μl of the sample to be extracted was added.
[0042] 3) The program on the NES-32 nucleic acid purifier was edited according to the steps shown in Table 3 and run:
TABLE-US-00003 TABLE 3 Nucleic acid extraction steps Magnetic Waiting Mixing attraction Hole time time time Mixing Volume Temperature Temperature Steps position Name (min) (min) (sec) speed (μl) state (° C.) 1 5 Transferring 0 1 60 Slow 100 Lysing 90 magnetic beads 2 1 Lysing 0 15 90 Slow 620 Lysing 90 3 2 Washing 1 0 2 90 Slow 600 Eluting 90 4 3 Washing 2 0 2 90 Slow 600 Eluting 90 5 4 Washing 3 0 0 90 Slow 600 Eluting 90 6 6 Eluting 0 5 90 Slow 100 Eluting 90 7 2 Discarding 0 1 0 Slow 600 Close 0 magnetic beads
[0043] 4) After completion of the automated extraction, the eluted products in columns 6 and 12 were transferred to a 1.5 ml centrifuge tube (a tip being inserted into the bottom of the tube) to obtain the extracted nucleic acid solution for use in subsequent experiments, or for later use by storage at −20° C. ±5° C.
Example 2: Steps of Fluorescent PCR Reaction
[0044] PCR reaction solution 1 for detecting HBV virus genotypes A and B and HBV DNA was prepared, the detection system being 40 μl. The detailed formulation of the PCR reaction solution 1 was as shown below:
[0045] PCR reaction solution 1: Tris-HCl (pH 8.9) 10 mmol/L, KCl 50 mmol/L, MgCl.sub.2 4 mmol/L, dNTPs each 150 umol/L, BSA 0.2 μg/μl, Primer1-F 100 nmol/L, Primer1-R 100 nmol/L, Probe1 75 nmol/L, Primer2-F 100 nmol/L, Primer2-R 100 nmol/L, Probe2 75 nmol/L, Primer3-F 100 nmol/L, Primer3-R 100 nmol/L, Probe3 75 nmol/L, Primer7-F 75 nmol/L, Primer7-R 75 nmol/L, and Probe7 50 nmol/L.
[0046] Enzyme mixture: Taq DNA polymerase 3.81 U/μl, UNG 0.224 U/μl, and anti-Taq DNA polymerase antibody 0.63 U/μl.
[0047] 19.5 μl of the PCR reaction solution 1 and 0.5 μl of the enzyme mixture were fully mixed, and then 20 μl of the resultant mixture was loaded into the first reaction tube and transferred to the sample processing region. 20 μl of the nucleic acid solution obtained in example 1 was added to the first reaction tube, then the first reaction tube was tightly covered and transferred to the detection region for real-time fluorescent PCR amplification and detection.
[0048] PCR reaction solution 2 for detecting HBV virus genotypes C and D and the C/D recombinant type was prepared, the detection system being 40 μl. The detailed formulation of the PCR reaction solution 2 was as shown below:
[0049] PCR reaction solution 2: Tris-HCl (pH 8.9) 10 mmol/L, KCl 50 mmol/L, MgCl.sub.2 4 mmol/L, dNTPs each 150 umol/L, BSA 0.2 μg/μl, Primer4-F 100 nmol/L, Primer4-R 100 nmol/L, Probe4 75 nmol/L, Primer5-F 100 nmol/L, Primer5-R 100 nmol/L, Probe5 75 nmol/L, Primer6-F 100 nmol/L, Primer6-R 100 nmol/L, Probe6 75 nmol/L, Primer7-F 75 nmol/L, Primer7-R 75 nmol/L, and Probe7 50 nmol/L;
[0050] enzyme mixture: Taq DNA polymerase 3.81 U/μl, UNG 0.224 U/μl, and anti-Taq DNA polymerase antibody 0.63 U/μl.
[0051] 19.5 μl of the PCR reaction solution 2 and 0.5 μl of the enzyme mixture were fully mixed, and then 20 μl of the resultant mixture was loaded into the second reaction tube and transferred to a sample processing region. 20 μl of the nucleic acid solution obtained in example 1 was added to the second reaction tube, then the second reaction tube was tightly covered and transferred to the detection region for real-time fluorescent PCR amplification and detection.
[0052] The real-time fluorescent PCR procedure was as shown in Table 4.
Example 4: Real-Time Fluorescent PCR Reaction Procedure
[0053]
TABLE-US-00004 Number Temperature Reaction Time Steps of cycles (° C.) (min:sec) 1 1 50 02:00 2 1 95 03:00 3 40 94 00:15 60 00:30
[0054] FAM, VIC, ROX and Cy5 were selected during detection, and data acquisition was set at 60° C.
[0055] Various channels and result explanation were as shown in Table 5.
TABLE-US-00005 TABLE 5 Various channels and result explanation Fluorescent Number channel setting Explanation PCR VIC Genotype A reaction FAM Genotype B solution 1 ROX HBV DNA Cy5 Reference gene PCR FAM Genotype C reaction VIC Genotype D, C/D solution 2 recombinant type ROX C/D recombinant type Cy5 Reference gene
[0056] 1. Internal Reference Determination
[0057] The Ct value of the reference gene channel (Cy5) in each reaction tube should be less than 40 in sample detection, and there was an obvious S-shaped amplification curve. If the above requirements were met, result determination was proceeded according to the following steps, otherwise the experiment was considered invalid, and errors in terms of instruments, reagents and amplification conditions should be checked.
[0058] 2. Determination of Detection Results
[0059] 2.1 Determination of HBV DNA: if the Ct value detected in the ROX channel of the PCR reaction solution 1 should be less than 40 and there was an obvious S-shaped amplification curve, it means that the sample was positive for HBV DNA, and then genotyping result determination was proceeded; and if there was no Ct value detected in the ROX channel of the PCR reaction solution 1, it means that the sample was negative for HBV DNA or below the lower limit of detection of reagents.
[0060] 2.2 HBV genotyping determination was as shown in Table 6.
TABLE-US-00006 TABLE 6 Determination results of HBV typing Reaction Fluorescent tube channel Detection results PCR reaction FAM − + − + − − solution 1 VIC + − − − − − PCR reaction FAM − − + + − − solution 2 VIC − − − − + + ROX / / / / − + Result determination Genotype Genotype Genotype Mixed Genotype C/D A B C B/C D recombinant type “−” indicates negative or below the lower limit of detection of the reagent, “+” indicates positive, “/” indicates no result determination, and “mixed B/C” indicates co-presence of genotype B and genotype C.
Example 3: Detection of Clinical Samples
[0061] 30 clinical samples were detected according to the methods in examples 1 and 2, and the detection results were as shown in Table 7.
TABLE-US-00007 TABLE 7 Detection Results of Clinical Samples Sample Number Detection Results of the Present invention Clinical Detected PCR Reaction solution 1 PCR Reaction solution 2 Detection Genes FAM VIC ROX CY5 FAM VIC ROX CY5 Results 1 + − + + − − / + B 2 + − + + − − / + B 3 + − + + − − / + B 4 − − + + − + − + D 5 + − + + − − / + B 6 − − + + + − / + C 7 + − + + − − / + B 8 + − + + − − / + B 9 + − + + − − / + B 10 + − + + + − / + Mixed B/C 11 − + + + − − / + A 12 + − + + + − / + Mixed B/C 13 − − + + − + + + C/D recombinant type 14 + − + + + − / + Mixed B/C 15 − − + + + − / + C 16 + − + + − − / + B 17 + − + + − − / + B 18 − − + + + − / + C 19 − − + + + − / + C 20 − − + + + − / + C 21 + − + + − − / + B 22 − − + + − + + + C/D recombinant type 23 − − + + − + + + C/D recombinant type 24 − − + + + − / + C 25 − − + + + − / + C 26 − − − + − − / + − 27 − − − + − − / + − 28 − − − + − − / + − 29 − − − + − − / + − 30 − − − + − − / + − Note: “+” indicates a positive result, “−” indicates negative or below the lower limit of detection of the reagent, and “/” indicates no result determination.
[0062] In these 30 samples, the results of the FAM, VIC, ROX and CY5 channels of the PCR reaction solution 1 were shown in
[0063] As shown in
Example 4: Detection Results of Sensitivity
[0064] HBV genotype A, B, C, D, and the C/D recombinant type positive samples were detected respectively according to the methods of Examples 1 and 2. Each positive sample was subjected to gradient dilution to a concentration of about 100 IU/ml, 50 IU/ml, 20 IU/ml, and 10 IU/ml, respectively and each concentration was detected repeatedly 8 times. The detection results were shown in Table 8 and
TABLE-US-00008 TABLE 8 Sensitivity detection results Genotype C/D recombinant Genotype A Genotype B Genotype C Genotype D type Sample Concentration Concentration Concentration Concentration Concentration (IU/ml) (IU/ml) (IU/ml) (IU/ml) (IU/ml) Repeat 100 50 20 10 100 50 20 10 100 50 20 10 100 50 20 10 100 50 20 10 1 + + + + + + + + + + + + + + + + + + + + 2 + + + + + + + + + + + + + + + + + + + + 3 + + + + + + + + + + + + + + + + + + + + 4 + + + + + + + + + + + + + + + + + + + + 5 + + + + + + + + + + + + + + + + + + + + 6 + + + + + + + + + + + + + + + + + + + + 7 + + + + + + + + + + + − + + + + + + + + 8 + + + + + + + + + + + + + + + − + + + + Note: “+” indicates a positive result, and “−” indicates negative or below the lower limit of detection of the reagent.
[0065] The experimental results showed that the detection sensitivity of the present invention was 10 IU/ml for genotype A, 10 IU/ml for genotype B, 20 IU/ml for genotype C, 20 IU/ml for genotype D, and 10 IU/ml for C/D recombinant type.