A PCR primer, a PCR kit and a method for detecting dengue virus by amplifying reverse transcription product of dengue virus

Abstract

This invention discloses PCR primers, a PCR kit, and a method for detecting dengue virus, belonging to the field of virus detection technology. It includes the upstream primer DENV-F as shown in SEQ ID NO.1 and the downstream primer DENV-R as shown in SEQ ID NO.2. This invention utilizes the high efficiency, speed, and accuracy of PCR reactions, the rapid speed of third-generation sequencing, the ability to provide approximately 10,000 bases of full-length sequencing data (each sequencing result representing a complete viral sequence), and the accuracy of phylogenetic analysis for strain analysis. It establishes a dengue virus detection method based on PCR amplification, third-generation sequencing, and phylogenetic analysis, playing a crucial role in the rapid and accurate detection of dengue virus. The PCR primers used in this invention have high sensitivity (down to 0.01 pg / reaction) and good specificity, amplifying dengue virus without reacting with other sequences.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, and in particular to a PCR primer for amplifying the reverse transcription product of dengue virus, a PCR kit, and a method for detecting dengue virus. Background Technology

[0002] Dengue virus (DENV) belongs to the genus Flaviviridae in the family Flaviviridae and is the pathogen that causes dengue fever in humans. Dengue fever is an acute infectious disease transmitted by Aedes mosquitoes, characterized by fever, generalized muscle and joint pain, and rash. Severe cases can lead to dengue hemorrhagic fever and dengue shock syndrome, with a high mortality rate. Dengue virus can be classified into four serotypes based on antigenicity. Humans who have been infected with dengue virus once are more likely to experience severe illness and shock when reinfected with a different serotype (i.e., a different serotype than the initial infection). Therefore, rapid and accurate testing of suspected patient samples is necessary, providing resolution at least up to the serotype level.

[0003] Currently, the gold standard for dengue virus detection is primarily based on antigen-antibody specific binding methods; the second is real-time quantitative PCR. These methods can currently determine whether suspected dengue patients are infected with the dengue virus, down to their serotype, but they cannot further determine the genotype or viral strain, nor can they accurately identify any high-risk mutations. Accurately obtaining information including strain and mutation details requires whole-genome sequencing of the dengue virus. Currently, whole-genome sequencing for dengue virus is generally based on next-generation sequencing (NGS), which is time-consuming, unsuitable for actual epidemic monitoring, and unable to identify multiple infections. Some laboratory studies have proposed using third-generation sequencing, but this requires first determining the dengue virus serotype and then using different primers for sequencing, which is inconvenient in practice and expensive. Therefore, providing a detection technology that can identify multiple infections is crucial. Summary of the Invention

[0004] The purpose of this invention is to provide PCR primers, a PCR kit, and a method for detecting dengue virus to amplify the reverse transcription product of dengue virus, thereby solving the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] One of the technical solutions of the present invention is a PCR primer for amplifying the reverse transcription product of dengue virus, comprising the upstream primer DENV-F as shown in SEQ ID NO.1 and the downstream primer DENV-R as shown in SEQ ID NO.2.

[0007] The second technical solution of the present invention is a PCR kit for detecting dengue virus, comprising the PCR primers.

[0008] The third technical solution of this invention is a one-step reverse transcription and amplification reaction system, comprising: 10 μL template, 2 μL amplification primers, and 25X SuperScript. TM IV RT Mix 0.8μL, SuperScript TM IV template switching oligonucleotide 1 μL, 2X UniPrime TM Add 10 μL of RT-PCRMaster Mix to a final volume of 20 μL with deionized water.

[0009] The fourth technical solution of this invention is a method for detecting dengue virus by reverse transcription, third-generation sequencing, and phylogenetic analysis for non-disease detection or treatment purposes, comprising the following steps:

[0010] (1) Using the RNA of the sample to be tested as a template, a one-step reverse transcription and amplification reaction is carried out using the reaction system to obtain the amplification product;

[0011] (2) Using the negative control as the control group, the amplification product is subjected to electrophoresis. If the amplification product has only one band and the length is about 10kb, and the negative control has no band, it can be determined that the sample to be tested contains dengue virus.

[0012] (3) Perform third-generation sequencing on the amplified products, and use a program to analyze the third-generation sequencing data based on the dengue virus phylogenetic tree, standard reference sequence, serotype, genotype and strain information to determine the phylogenetic position of the sample and its related strains.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] This invention utilizes the high efficiency, speed, and accuracy of PCR, the rapid speed of third-generation sequencing (which provides approximately 10,000 bases of full-length sequencing data, with each sequencing result representing a complete viral sequence), and the accuracy of phylogenetic analysis for strain analysis. It establishes a dengue virus detection method based on PCR amplification, third-generation sequencing, and phylogenetic analysis, playing a crucial role in the rapid and accurate detection of dengue virus. The PCR primers used in this invention exhibit high sensitivity (down to 0.01 pg / reaction) and good specificity, amplifying dengue virus without reacting with other sequences. This invention can obtain information such as genotype, strain, mutation, and evolution that traditional detection methods cannot provide, which is of great significance for identifying dengue virus-infected strains and provides a foundation for precise dengue fever prevention and control and precision medicine.

[0015] This invention, without requiring specific dengue virus serotypes, rapidly obtains information including serotype, genotype, strain, mutation, and phylogenetic data, which is unattainable by traditional detection methods such as antigen-antibody binding assays and RT-qPCR. It also avoids the problems of long processing times and inability to identify multiple infections associated with next-generation whole-genome sequencing. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This study describes a method for detecting dengue virus using reverse transcription, third-generation sequencing, and phylogenetic analysis.

[0018] Figure 2 This is a one-step reverse transcription-amplification system for detection from low starting RNA samples. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0025] This invention provides PCR primers for amplifying the reverse transcription product of dengue virus, including the upstream primer DENV-F as shown in SEQ ID NO.1 and the downstream primer DENV-R as shown in SEQ ID NO.2.

[0026] This invention also provides a PCR kit for detecting dengue virus, including the PCR primers.

[0027] This invention also provides a one-step reverse transcription and amplification reaction system, comprising: 10 μL template, 2 μL amplification primers, and 25X SuperScript. TM IV RT Mix 0.8μL, SuperScript TM IV template switching oligonucleotide 1 μL, 2X UniPrime TM Add 10 μL of RT-PCRMaster Mix to a final volume of 20 μL with deionized water.

[0028] This invention also provides a method for detecting dengue virus by reverse transcription, third-generation sequencing, and phylogenetic analysis for purposes other than disease detection or treatment, comprising the following steps:

[0029] (1) Using the RNA of the sample to be tested as a template, a one-step reverse transcription and amplification reaction is carried out using the reaction system to obtain the amplification product;

[0030] (2) Using the negative control as the control group, the amplification product is subjected to electrophoresis. If the amplification product has only one band and the length is about 10kb, and the negative control has no band, it can be determined that the sample to be tested contains dengue virus.

[0031] (3) Perform third-generation sequencing on the amplified products, and use a program to analyze the third-generation sequencing data based on the dengue virus phylogenetic tree, standard reference sequence, serotype, genotype and strain information to determine the phylogenetic position of the sample and its related strains.

[0032] In some specific implementations, the reaction program for the one-step reverse transcription and amplification is 50℃ for 10 min; 98℃ for 2 min; 98℃ for 10 s, 60℃ for 10 s, 72℃ for 5.5 min, for 35 cycles; 72℃ for 5 min.

[0033] In some specific implementations, the third-generation sequencing method includes the following steps:

[0034] (1) Using Beckman TM AMPure XP Beads and magnetic racks were used to purify the amplification products;

[0035] (2) Using Nanopore TM The Native Barcoding Kit 24v14 performs tailing, barcoding, and sequencing adapter addition on the purified product obtained in step (1).

[0036] (3) Using Nanopore TM Sequencing was performed using a MinION flow cell R10.4, and the generated files were output by Dorado in fastq format for downstream analysis.

[0037] In some specific implementations, the method for analyzing third-generation sequencing data based on dengue virus phylogenetic tree, standard reference sequence, serotype, genotype, and strain information includes the following steps:

[0038] (1) Use the fastp program to filter the fastq format file;

[0039] (2) Use SAMTools and Minimap2 in conjunction with a standard reference sequence to perform sequence alignment on the filtering results obtained in step (1);

[0040] (3) Use the analysis program developed in conjunction with the TIPars program to identify the phylogenetic position of the sequence obtained in step (2) on the phylogenetic tree;

[0041] (4) Output the relevant phylogenetic information of the sample sequence based on the serotype, genotype, and evolutionary position of the reference sequence that is closest to the sample sequence in phylogenetic position;

[0042] (5) Based on the filtering results, the mutations that the dengue virus may carry in the sample can be directly analyzed by comparing the output sequencing results with the standard reference sequence.

[0043] Example 1

[0044] 1. Specific primers designed targeting the conserved regions of the coding gene of dengue virus

[0045] A total of 1,500 dengue virus sequences, including serotypes 1-4, were downloaded from the NCBI nucleotide database, complete and free of ambiguous bases. Based on the alignment results, a pair of specific primers, DENV-F / DENV-R, was designed and screened to amplify all four serotypes of dengue virus, targeting highly conserved fragments in the coding region. The target fragment length of dengue virus amplified was 9,990 bases.

[0046] The primer sequences are as follows:

[0047] DENV-F: 5'-TGCTGAAACGCGAGAGAAAACCG-3' (SEQ ID NO. 1);

[0048] DENV-R: 5'-AGTGATCCGCACCATTGGTCTT-3' (SEQ ID NO. 2).

[0049] 2. Extract RNA and perform reverse transcription and amplification.

[0050] (1) The RNA extraction system includes: SuperScript TM IV 10X lysis buffer 1 μL, SuperScript TM IV capture oligonucleotide 1 μL, RNase inhibitor 0.4 μL, and test sample 7.6 μL. Mix by pipetting 8 times, then centrifuge rapidly, followed by incubation at 72°C for 1 minute, and then centrifuge again.

[0051] (2) The reaction system for one-step reverse transcription amplification includes: approximately 10 μL of the product obtained in the previous step, 1 μL of amplification primer DENV-F, 1 μL of amplification primer DENV-R, and 25X SuperScript. TM IV RT Mix 0.8μL, SuperScript TM IV template switching oligonucleotide 1 μL, 2X UniPrime TM 10 μL of RT-PCR Master Mix was added to a final volume of 20 μL with deionized water. One-step reverse transcription and amplification were performed using a PCR instrument. The reaction program was: 50℃ for 10 min; 98℃ for 2 min; 98℃ for 10 s, 60℃ for 10 s, 72℃ for 5.5 min, for 35 cycles; 72℃ for 5 min.

[0052] (4) After amplification, amplification detection is performed. The detection method is as follows: the same operation is performed on the negative control using a PCR DNA ladder during product preparation. If electrophoresis after amplification shows that the amplified product has only one band and is close to the 10kb position of the PCR DNA ladder, while the negative control has no band, it can be determined that the product has been successfully and correctly amplified, the sample contains dengue virus, and the patient is confirmed to be infected with dengue fever. The negative control is the recombinant plasmid pET-30a without any inserted fragments.

[0053] 3. Processing and sequencing of the amplification products

[0054] (1) Using Beckman TM The amplification products were purified using AMPure XPBeads and magnetic racks according to their instructions.

[0055] (2) Using Nanopore TM The Native Barcoding Kit 24v14 kit was used to perform tailing, barcode addition, and sequencing adapter addition on the purified product obtained in step (1) according to its instructions.

[0056] (3) Using Nanopore TM Sequencing was performed using a MinION flow cell R10.4, and the generated files were output by Dorado in fastq format for downstream analysis.

[0057] 4. Analyze the sequencing results to determine the phylogenetic position of the virus.

[0058] (1) Use the sequences downloaded from the NCBI nucleotide database in step 1 to construct a phylogenetic tree and a standard reference sequence, and enter information such as serotype, genotype, and high-risk strains;

[0059] (2) Use the fastp program to filter the sequencing results in fastq format obtained in step 3(3);

[0060] (3) Use SAMTools and Minimap2 in combination with the standard reference sequence obtained in step (1) to align the result obtained in step (2);

[0061] (4) Use the analysis program developed in conjunction with the TIPars program to identify the phylogenetic position of the sequence obtained in step (3) on the phylogenetic tree;

[0062] (5) Based on the results of step (4), output the relevant phylogenetic information of the sample sequence according to the serotype, genotype and evolutionary position of the reference sequence that is closest to the sample sequence;

[0063] (6) Based on the results obtained in step (2), the mutations that dengue virus may carry in the sample can be directly analyzed by comparing the output sequencing results with the standard reference sequence in step (1).

[0064] Example 2

[0065] 1. In the sensitivity test, the reaction system of the one-step reverse transcription amplification method was tested. The results showed that the sensitivity of this method can reach 0.01 pg / reaction ( Figure 2 It has good sensitivity.

[0066] 2 Specificity

[0067] Using other viruses from the Flaviviridae family, which are phylogenetically closest to dengue virus, including Japanese encephalitis virus (TaxID: 11072), tick-borne encephalitis virus (TaxID: 11084), and hepatitis C virus (TaxID: 3052230), the primers were found to be unable to amplify them. This result demonstrates that the designed specific primers DENV-F / DENV-R, the kits designed based on them, and the detection method established based on them possess good specificity.

[0068] 3 Repeatability

[0069] For dengue virus samples with full-length genome information collected in Guangzhou in 2023, a dengue virus serotype 1 gene type I sequence and a dengue virus serotype 2 gene Cosmopolitan sequence were used six times. It was found that the corresponding serotype, genotype and strain could be found on the phylogenetic tree formed by the amplified region of both sequences, proving that the method is reproducible.

[0070] 4. Accuracy

[0071] Analysis of the primer amplification region of this invention using 70 sequences from Guangzhou revealed that all sequences could be correctly predicted for their serotype, genotype, and even genotype subtype. Five sequences—three from serotype 1, one from serotype 2, and one from serotype 3—failed to be assigned to the exact phylogenetic position predicted by the whole-genome phylogenetic tree, but remained within their corresponding lines. Therefore, the accuracy of this method is high and far exceeds the precision and resolution of traditional methods (which are only accurate to serotype). It was noted that 1.0%–4.0% of sequences after nanopore third-generation sequencing failed to be assigned to the same phylogenetic position as the remaining sequences. This was due to sequencing errors rather than primer or kit issues. Excluding multiple infections, this method only considers and reports the phylogenetic information of >20% of sequences assigned to the same position in each sample.

[0072] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting dengue virus by reverse transcription, third-generation sequencing, and phylogenetic analysis for non-disease detection or treatment purposes, characterized in that, Includes the following steps: (1) Using RNA from the sample to be tested as a template, a one-step reverse transcription and amplification reaction is performed using the reaction system to obtain the amplification product; the reaction system includes: 10 μL template, 2 µL amplification primers, 0.8 µL 25X SuperScript™ IV RT Mix, 1 µL SuperScript™ IV template switching oligonucleotide, 10 µL 2X UniPrime™ RT-PCR Master Mix, and deionized water to 20 μL; the amplification primers include the upstream primer DENV-F as shown in SEQ ID NO.1 and the downstream primer DENV-R as shown in SEQ ID NO.2; (2) Using the negative control as the control group, the amplification product is subjected to electrophoresis. If the amplification product has only one band and the length is about 10kb, and the negative control has no band, it can be determined that the sample to be tested contains dengue virus. (3) Perform third-generation sequencing on the amplified products, and use a program to analyze the third-generation sequencing data based on the dengue virus phylogenetic tree, standard reference sequence, serotype, genotype and strain information to determine the phylogenetic position of the sample and its related strains.

2. The method according to claim 1, characterized in that, The reaction program for the one-step reverse transcription and amplification is as follows: 50℃ for 10 min; 98℃ for 2 min; 98℃ for 10 s, 60℃ for 10 s, 72℃ for 5.5 min, for 35 cycles; 72℃ for 5 min.

3. The method of claim 1, wherein, The third-generation sequencing method includes the following steps: (1) The amplification products were purified using Beckman™ AMPure XP Beads and a magnetic rack; (2) The purified product obtained in step (1) was processed by tailing, barcoding, and sequencing adapter addition using the Nanopore™ Native Barcoding Kit 24 v14. (3) Sequencing was performed using the Nanopore™ MinION flow cell R10.

4. The generated files were output by Dorado in fastq format and could be used for downstream analysis.

4. The method of claim 3, wherein, The method for analyzing third-generation sequencing data based on dengue virus phylogenetic tree, standard reference sequence, serotype, genotype, and strain information includes the following steps: (1) Use the fastp program to filter the fastq format file as described in claim 3; (2) Use SAMTools and Minimap2 in conjunction with a standard reference sequence to perform sequence alignment on the filtering results obtained in step (1); (3) Use the analysis program developed in conjunction with the TIPars program to identify the phylogenetic position of the sequence obtained in step (2) on the phylogenetic tree; (4) Output the relevant phylogenetic information of the sample sequence based on the serotype, genotype, and evolutionary position of the reference sequence that is closest to the sample sequence in phylogenetic position; (5) Based on the filtering results, the mutations that the dengue virus may carry in the sample can be directly analyzed by comparing the output sequencing results with the standard reference sequence.

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