PCR (Polymerase Chain Reaction) primer for amplifying reverse transcription product of dengue virus, PCR kit and method for detecting dengue virus

Abstract

The invention discloses a PCR (Polymerase Chain Reaction) primer for amplifying a reverse transcription product of dengue virus, a PCR kit and a method for detecting the dengue virus, and belongs to the technical field of virus detection. Comprising an upstream primer DENV-F as shown in SEQ ID NO. 1 and a downstream primer DENV-R as shown in SEQ ID NO. 2. The dengue virus detection method based on the PCR amplification reaction, the three-generation sequencing and the phylogenetic analysis is established by utilizing the characteristics that the PCR reaction is efficient, rapid and accurate, the three-generation sequencing is rapid, the full-length sequencing of about 10,000 basic groups can be provided, one sequencing result represents one complete virus sequence, the phylogenetic analysis is accurate in strain analysis and the like. The kit plays a role in rapid and accurate detection of dengue viruses. The PCR primer used in the invention has high sensitivity, can reach 0.01 pg / reaction, has good specificity, and does not react with other sequences while amplifying the dengue virus.

Description

Technical Field

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

[0002] Dengue virus (DENV) belongs to the genus Flavivirus of the family Flaviviridae and is the pathogen that causes dengue fever in humans. Dengue fever is an acute infectious disease transmitted by the bites of Aedes mosquitoes, with fever, systemic muscle and bone and joint pain, and rash as the main symptoms. Severe patients will develop dengue hemorrhagic fever and dengue shock syndrome, with a high mortality rate. Dengue viruses can be divided into four serotypes based on antigenicity. Humans who have been infected with dengue virus once will have a more severe condition and are more likely to suffer shock when they are infected with a different type of dengue virus (i.e., the serotype is different from the first time). Therefore, it is necessary to be fast and accurate when testing samples from suspected patients, and to provide a resolution of at least the serotype.

[0003] At present, the gold standard for dengue virus detection is mainly based on the method of antigen-antibody specific binding; the second is the use of real-time fluorescence quantitative PCR for detection. This method can currently determine whether a suspected dengue patient is infected with dengue virus, accurately to its serotype, but cannot further determine its genotype and virus strain, nor can it accurately point out the high-risk mutations it may carry. Accurately obtaining information including strains and mutations requires whole genome sequencing of dengue virus. At present, whole genome sequencing of dengue virus is generally based on second-generation sequencing, which is time-consuming, not conducive to actual epidemic monitoring, and cannot identify multiple infections; some laboratory studies have proposed the use of third-generation sequencing, but its sequencing requires first determining the dengue virus serotype and then using different primers for sequencing, which is inconvenient in actual operation and expensive. Therefore, it is crucial to provide a detection technology that can identify multiple infections. Summary of the invention

[0004] The purpose of the present invention is to provide a PCR primer for amplifying the reverse transcription product of dengue virus, a PCR kit and a method for detecting dengue virus, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is a PCR primer for amplifying the reverse transcription product of dengue virus, comprising an upstream primer DENV-F as shown in SEQ ID NO.1 and a 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 the present invention is a one-step reverse transcription and amplification reaction system, comprising: 10 μL of template, 2 μL of amplification primer, 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, add deionized water to 20 μL.

[0009] A fourth technical solution of the present 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, performing a one-step reverse transcription and amplification reaction using the reaction system to obtain an amplification product;

[0011] (2) using the negative control as the control group, performing electrophoresis on the amplified product. If the amplified product has only one band with a length of about 10 kb and the negative control has no band, it can be determined that the sample to be tested contains dengue virus;

[0012] (3) performing third-generation sequencing on the amplified product, and using 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] The present invention utilizes the characteristics of high efficiency, rapidity and accuracy of PCR reaction, rapidity of third-generation sequencing and the ability to provide full-length sequencing of about 10,000 bases and one sequencing result represents a complete virus sequence, and accurate analysis of strains by phylogenetic analysis, etc., to establish a dengue virus detection method based on PCR amplification reaction, third-generation sequencing, and phylogenetic analysis, which plays a role in the rapid and accurate detection of dengue virus. The PCR primers used in the present invention have high sensitivity, which can reach 0.01pg / reaction, good specificity, and will not react with other sequences while amplifying dengue virus. The present invention can obtain information that cannot be obtained by traditional detection methods, such as genotype, strain, mutation, evolution, etc., which is of great significance for determining the strain infected with dengue virus, and provides a basis for accurate prevention and control of dengue fever and precision medicine.

[0015] The present invention does not require the dengue virus serotype to be specified without spending much time, and can quickly obtain information including serotype, genotype, strain, mutation, phylogeny, etc., which are unattainable by traditional detection schemes such as antigen-antibody binding detection and RT-qPCR detection. It can also avoid the problems of long time spent on second-generation whole genome sequencing and inability to identify multiple infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 A method for detecting dengue virus through reverse transcription, third-generation sequencing and phylogenetic analysis.

[0018] Figure 2 It is a one-step reverse transcription-amplification system for detection from low input RNA samples. DETAILED DESCRIPTION

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

[0020] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

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

[0024] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

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

[0026] The embodiment of the present invention also provides a PCR kit for detecting dengue virus, comprising the PCR primers.

[0027] The embodiment of the present invention also provides a one-step reverse transcription and amplification reaction system, including: 10 μL of template, 2 μL of amplification primer, 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, add deionized water to 20 μL.

[0028] The embodiment of the present invention also provides 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:

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

[0030] (2) using the negative control as the control group, performing electrophoresis on the amplified product. If the amplified product has only one band with a length of about 10 kb and the negative control has no band, it can be determined that the sample to be tested contains dengue virus;

[0031] (3) performing third-generation sequencing on the amplified product, and using 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 embodiments, the reaction procedure of the one-step reverse transcription and amplification is 50°C for 10 min; 98°C for 2 min; 98°C for 10 s, 60°C for 10 s, 72°C for 5.5 min, 35 cycles; 72°C for 5 min.

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

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

[0035] (2) Using Nanopore TM The Native Barcoding Kit 24v14 kit performs tail-end processing, barcoding, and sequencing adapter addition operations on the purified product obtained in step (1);

[0036] (3) Using Nanopore TM The sequencing was performed using MinION flow cell R10.4, and the files generated after sequencing were exported by Dorado into fastq format files that can be used for downstream analysis.

[0037] In some specific embodiments, the method of using a program to analyze third-generation sequencing data based on a dengue virus phylogenetic tree, a standard reference sequence, serotype, genotype, and strain information comprises the following steps:

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

[0039] (2) Use SAMTools and Minimap2 combined with standard reference sequences to align the filtered results obtained in step (1);

[0040] (3) using an analysis program developed in conjunction with the TIPars program to identify the sequence obtained in step (2) and find its phylogenetic position on the phylogenetic tree;

[0041] (4) Output 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 based on the output sequencing results and the standard reference sequence.

[0043] Example 1

[0044] 1 Specific primers designed for the conservative region of the coding region gene of dengue virus

[0045] A total of 1,500 complete dengue virus sequences with no ambiguous bases, including serotypes 1-4, were downloaded from the NCBI nucleotide database. Based on the alignment results, a pair of specific primers DENV-F / DENV-R that can amplify all four serotypes of dengue virus were designed and screened for the highly conserved fragments in the coding region. The target fragment length of the amplified dengue virus 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) RNA extraction system includes: SuperScript TM IV 10X Lysis Buffer 1 μL, SuperScript TM 1 μL IV capture oligonucleotide, 0.4 μL RNase inhibitor, and 7.6 μL sample to be tested. Pipette 8 times to mix, then centrifuge quickly, then place in a 72°C water bath for 1 minute, and centrifuge quickly;

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

[0052] (4) After amplification, amplification detection is performed. The detection method is: when preparing the product, use PCR DNA ladder to perform the same operation on the negative control. If electrophoresis is performed after amplification and it is found that the amplified product has only one band and is close to the 10kb position of PCR DNA ladder, and the negative control has no band, it can be judged that the product has been successfully 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 fragment inserted.

[0053] 3. Processing and sequencing of amplified products

[0054] (1) Using Beckman TM AMPure XPBeads and magnetic rack were used to purify the amplified products according to their instructions;

[0055] (2) Using Nanopore TM The purified product obtained in step (1) was subjected to tail-end treatment, barcode ligation, and sequencing adapter ligation using the Native Barcoding Kit 24v14 according to the instructions.

[0056] (3) Using Nanopore TM The sequencing was performed using MinION flow cell R10.4, and the files generated after sequencing were exported by Dorado into fastq format files that can be used 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 standard reference sequence, and enter information such as serotype, genotype, and high-risk strains;

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

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

[0061] (4) using an analysis program developed in conjunction with the TIPars program to identify the sequence obtained in step (3) and find its phylogenetic position 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 closest to the sample sequence;

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

[0064] Example 2

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

[0066] 2 Specificity

[0067] Other viruses in the Flaviviridae family, which are phylogenetically closest to dengue virus, including Japanese encephalitis virus (TaxID: 11072), Tick-borne encephalitis virus (TaxID: 11084), and Hepacivirus hominis (TaxID: 3052230), were found to be unable to be amplified by the primers. This result indicates that the designed specific primers DENV-F / DENV-R, the kit designed based on them, and the detection method established based on them have good specificity.

[0068] 3. Repeatability

[0069] For the dengue virus collected in Guangzhou in 2023 and already with full-length genome information, a dengue virus serotype 1 gene type I sequence and a dengue virus serotype 2 gene Cosmopolitan type sequence were used repeatedly 6 times. It was found that the two sequences could retrieve the corresponding serotype, genotype, and strain on the phylogenetic tree formed in the amplified region, proving that this method is repeatable.

[0070] 4. Accuracy

[0071] 70 sequences from Guangzhou were used to analyze the amplified regions of the invention primers, and it was found that all sequences could be correctly predicted for their serotype, genotype, and even genotype; 3 sequences from serotype 1, 1 sequence from serotype 2, and 1 sequence from serotype 3, a total of 5 sequences, failed to be assigned to the same phylogenetic position as expected in the whole genome phylogenetic tree, but were still in the corresponding strains, so the accuracy of this method is high and has completely exceeded the accuracy and resolution of traditional methods (which can only be accurate to serotypes). It is noted that after nanopore third-generation sequencing, 1.0%-4.0% of the sequences failed to be assigned to the same phylogenetic position as the remaining sequences. This is caused by sequencing errors rather than primers or kits. In the case of excluding multiple infections, this method only considers and reports the phylogenetic information of sequences that are >20% 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 methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A PCR primer for amplifying a reverse transcription product of dengue virus, characterized in that: It includes an upstream primer DENV-F as shown in SEQ ID NO.1 and a downstream primer DENV-R as shown in SEQ ID NO.

2.

2. A PCR kit for detecting dengue virus, characterized in that: Comprising the PCR primers according to claim 1.

3. A one-step reverse transcription and amplification reaction system, characterized in that: include: Template 10μL, amplification primer 2μL, 25XSuperScript TM IV RT Mix 0.8μL, SuperScript TM IV Template Switching Oligonucleotide 1 μL, 2X UniPrime TM 10 μL of RT-PCR Master Mix, add deionized water to 20 μL.

4. 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: The following steps are involved: (1) using the RNA of the sample to be tested as a template, performing a one-step reverse transcription and amplification reaction using the reaction system described in claim 3 to obtain an amplification product; (2) using the negative control as the control group, performing electrophoresis on the amplified product. If the amplified product has only one band with a length of about 10 kb and the negative control has no band, it can be determined that the sample to be tested contains dengue virus; (3) performing third-generation sequencing on the amplified product, and using 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.

5. The method according to claim 4, characterized in that The reaction procedure of the one-step reverse transcription and amplification is 50°C for 10 min; 98°C for 2 min; 98°C for 10 s, 60°C for 10 s, 72°C for 5.5 min, 35 cycles; 72°C for 5 min.

6. The method according to claim 4, characterized in that The method for the third generation sequencing comprises the following steps: (1) Using Beckman TM AMPure XP Beads and magnetic rack were used to purify the amplified products; (2) Using Nanopore TM The Native Barcoding Kit 24v14 kit performs tail-end processing, barcoding, and sequencing adapter addition operations on the purified product obtained in step (1); (3) Using Nanopore TM The sequencing was performed using MinION flow cell R10.4, and the files generated after sequencing were exported by Dorado into fastq format files that can be used for downstream analysis.

7. The method according to claim 4, characterized in that The method for analyzing the third generation sequencing data using the program based on the dengue virus phylogenetic tree, standard reference sequence, serotype, genotype and strain information comprises the following steps: (1) using the fastp program to filter the fastq format file described in claim 6; (2) Use SAMTools and Minimap2 combined with standard reference sequences to align the filtered results obtained in step (1); (3) using an analysis program developed in conjunction with the TIPars program to identify the sequence obtained in step (2) and find its phylogenetic position on the phylogenetic tree; (4) Output 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 based on the output sequencing results and the standard reference sequence.

Images