WO2017185244A1 - Rt-pcr-hrm or pcr-hrm primer, reagent, and method for rapidly distinguishing four types of serum dengue virus - Google Patents

Abstract

The present invention discloses an RT-PCR-HRM or PCR-HRM primer, reagent, and method for rapidly distinguishing four types of serum dengue virus.

Description

 RT-PCR-HRM or PCR-HRM primer, reagent and method for rapidly distinguishing four serotypes of dengue virus

Technical field

The invention relates to a method for virus genotyping, in particular to an RT-PCR-HRM or PCR-HRM primer and method for rapidly distinguishing four serotypes of dengue virus.

Background technique

Dengue virus Virus, DV) is a single-stranded positive-strand RNA virus with envelope. It belongs to the flavivirus of the Flaviviridae virus. Its main vectors are Aedes aegypti and Aedes albopictus. In China, it is mainly Aedes albopictus. It is causing human dengue fever (dengue Fever, DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (dengue shock) The main pathogen of syndrome, DSS). In 1779, in Jakarta, Indonesia, Bylon first described this disease called joint fever. In 1869, the Royal College of Internal Medicine of London named the disease dengue fever. Dengue fever and dengue hemorrhagic fever are widely prevalent in more than 60 countries and regions in the tropical and subtropical regions of the world, especially in Southeast Asia, the Pacific Islands and the Caribbean. In Europe and North America, small-scale infections caused by imported cases have also occurred. . In the 20th century, dengue fever has occurred in many pandemics around the world, with millions of cases. About 2.5 billion people worldwide are threatened by dengue virus infection. About 50 million to 100 million people are infected with dengue virus each year, 500,000. People need to be admitted to hospital for treatment, and about 24,000 people die. In the popular areas of China, there are Guangdong, Guangxi, Hainan, Liaoning and other places. The popular season is Hainan from April to June, and Guangdong and Guangxi are August. As early as 1873, Dengue fever occurred in Xiamen, China. From 1928 to 1929, it was popular in Guangzhou, Xiamen, Hangzhou, Ningbo, Shanghai, Taiwan and Hong Kong. In 1940, the disease was prevalent in Shanghai and Nantong. From 1942 to 1945, the epidemic became more serious, not only popular in coastal areas, but even spread to the mainland such as Hankou and other places. In May 1978, dengue fever occurred in Shiwan Town, Foshan City, Guangdong Province, and the epidemic spread rapidly. In the summer of 2002, there was a dengue fever epidemic in Guangzhou, with more than 1,000 people developing the disease. Since June 2014, a large-scale dengue epidemic has occurred in Guangdong Province. As of mid-November, the incidence of dengue fever in Guangdong Province has exceeded 44,000 and 6 deaths have been reported. In Kaohsiung City, Taiwan, as of mid-November 2014, the annual incidence of dengue fever exceeded 10,000 cases and 15 deaths were reported. From 2015 to November 3, Taiwan Province reported a total of 29,921 cases of dengue fever, including 21,874 cases in Tainan City, 7521 cases in Kaohsiung City, and 151 cases in Pingtung County. The rest of the counties and cities were affected by the disease. The number of people was 129, and 27 suspected deaths were pending (9 in Tainan, 17 in Kaohsiung, and 1 in Pingtung County). There are still 28 people in Taiwan who are in the intensive care unit. In mainland China, as of October 17, 2015, a total of 1,273 cases of dengue fever were reported in Chaozhou, Guangdong.

The above data show that in recent years, dengue fever has been experiencing widespread epidemics in subtropical and tropical regions of China, causing serious social public health problems in the local area.

Dengue virus can be divided into four antigenic closely related serotypes, namely dengue 1, 2, 3, and 4. The nucleotide sequence homology between each type of dengue virus is 63%-68%, and the nucleotide sequence homology between the virus strains of the same serotype is greater than 95%. Four serotypes can cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome. DV can present two different states after infection of the body, namely the primary infection state and the secondary infection state. After the first infection of dengue virus, the body may present as a latent infection or general fever, the symptoms are not obvious, and at the same time, it is immune to the same type of virus, and may last for a lifetime, but the protection against heterotypic virus infection lasts for a short time. DHF and DSS are prone to occur in patients with a second infection with atypical DV, and the mortality rate is high, and the pathogenesis is dependent on antibody-enhanced infection. Distinguishing between these two states is very important for clinical treatment. Since there are no successful vaccines available and there are no specific treatments, the early diagnosis and classification of dengue virus infections is very important for clinical treatment and disease monitoring, epidemiological investigations and disease control measures. Significance.

In the laboratory diagnostic methods for dengue virus infection, serological tests and molecular biology research have made some progress. The identification and classification of dengue virus, the traditional methods mainly include virus isolation and identification, hemagglutination inhibition test, complement binding test, neutralization test and so on. Sensitive cell culture or animal isolation viruses remain the "gold standard" for diagnosis, but this method is time consuming and many laboratories lack the conditions necessary to carry out virus isolation. On the other hand, virus-specific antibodies can be detected after 3 to 5 days of onset, and the early detection sensitivity of serology is not high, and the specificity is poor, the operation is cumbersome, time-consuming, and especially difficult to accurately classify. With the successful development of various types of dengue virus monoclonal antibodies, Classification methods based on dengue virus-type monoclonal antibodies, such as indirect immunofluorescence (IFA) and enzyme-linked immunosorbent assay (ELISA), are widely used in various dengue virus testing laboratories around the world. The problem of accurate typing of dengue virus has been solved, but there are still problems with cross-reaction with other flaviviruses, which are time consuming, costly and complicated to operate. In 1990, Deubel et al first reported the use of PCR technology to detect dengue virus nucleic acid. In 1992, Lanciotti et al. designed a nested PCR primer that was typed using different dengue virus PCR amplification products. In the following ten years, there have been many reports on the application of PCR technology to detect dengue virus in Chinese and foreign literatures, from multi-tube two-step method to single-tube one-step method, from four pairs of specific primers to single tube multiples. Reaction, but there are still many problems such as the need for multiple pairs of primers, electrophoresis detection links are easily contaminated, resulting in false positives. Although fluorescent PCR is not easily contaminated, it requires a large number of primer probes, which are expensive and thus cannot be widely used.

None of the above methods overcome the disadvantages of complicated operation, time consuming, high cost, and poor reliability, and the application in clinical practice is limited. Therefore, there is an urgent need for a dengue virus serotype typing method which is relatively simple in operation, reliable in detection results, and low in detection cost.

Summary of the invention

In order to solve the above problems, the present invention establishes an RT-PCR-HRM or PCR-HRM primer and method for rapidly distinguishing different serotypes of dengue virus, which is simple, rapid, reliable, and low in detection cost. Conducive to the promotion of applications in clinical practice.

It is an object of the present invention to provide three sets of RT-PCR-HRM or PCR-HRM primers that rapidly distinguish between different serotypes of dengue virus.

Another object of the present invention is to provide an RT-PCR-HRM or PCR-HRM method for rapidly distinguishing different serotypes of dengue virus.

It is still another object of the present invention to provide an RT-PCR-HRM or PCR-HRM reagent for rapidly distinguishing different serotypes of dengue virus.

The technical solution adopted by the present invention is:

The use of base sites for rapid differentiation of different serotypes of dengue virus, the base sites are:

The Genbank number is the 10648th base G in the genome of the DV1 standard strain of EU848545.1, the 10671th base T, the 10672th base A, the 10675th base G;

Genbank number is AF038403.1 The DV2 standard strain genome has the 10638th base A, the 10661th base C, the 10662th base T, the 10665th base G;

The Genbank number is the 106th base G of the DV3 standard strain genome of M93130.1, the 10633th base C, the 10634th base T, the 10637th base G;

Genbank number is the 10570th base A in the genome of the DV4 standard strain of AY947539.1, the base T of the 10593th base, the base G of the 10594th base, and the base A of the 10597th.

The use of gene fragments to rapidly distinguish between different serotypes of dengue viruses, which are:

67bp gene fragment:

5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCTACAGCATCATTCCAGGCACAG-3' (SEQ ID NO: 171);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’ (SEQ ID NO: 172);

5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’ (SEQ ID NO: 173);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTACAACATCAATCCAGGCACAG-3’ (SEQ ID NO: 174);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAG-3’ (SEQ ID NO: 175);

45bp gene fragment:

5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 176);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 177);

5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 178);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 179);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 180).

An RT-PCR-HRM or PCR-HRM primer for rapidly distinguishing different serotypes of dengue virus, the target gene fragment amplified by the primer contains the following base sites:

The Genbank number is the 10648th base G in the genome of the DV1 standard strain of EU848545.1, the 10671th base T, the 10672th base A, the 10675th base G;

Genbank number is AF038403.1 The DV2 standard strain genome has the 10638th base A, the 10661th base C, the 10662th base T, the 10665th base G;

The Genbank number is the 106th base G of the DV3 standard strain genome of M93130.1, the 10633th base C, the 10634th base T, the 10637th base G;

Genbank number is the 10570th base A in the genome of the DV4 standard strain of AY947539.1, the base T of the 10593th base, the base G of the 10594th base, and the base A of the 10597th.

An RT-PCR-HRM or PCR-HRM primer for rapidly distinguishing different serotypes of dengue virus, the primers comprising primers P1, P2 and P3;

The base sequence of the primer P1 is selected from at least one of SEQ ID NOS: 1-68;

The base sequence of the primer P2 is selected from at least one of SEQ ID NOS: 69 to 96;

The base sequence of the primer P3 is selected from at least one of SEQ ID NOS: 97 to 170.

An RT-PCR-HRM or PCR-HRM primer for rapidly distinguishing different serotypes of dengue virus, the primers comprising primers P1, P2 and P3;

The base sequence of the primer P1 is selected from at least one of SEQ ID NOS: 1 to 20;

The base sequence of the primer P2 is selected from at least one of SEQ ID NOS: 69 to 80;

The base sequence of the primer P3 is selected from at least one of SEQ ID NOS: 97 to 120.

An RT-PCR-HRM or PCR-HRM primer for rapidly distinguishing different serotypes of dengue virus, the primers comprising primers P1, P2 and P3;

The base sequence of the primer P1 is selected from at least one of SEQ ID NOS: 1 to 4;

The base sequence of the primer P2 is selected from at least one of SEQ ID NOS: 69 to 71;

The base sequence of the primer P3 is selected from at least one of SEQ ID NOS: 99 to 101.

A RT-PCR-HRM or PCR-HRM reagent for rapidly distinguishing different serotypes of dengue virus, the reagents containing the primers P1, P2 and P3 described above.

An RT-PCR-HRM or PCR-HRM method for rapidly distinguishing different serotypes of dengue virus, comprising the following steps:

1) Preparation of a template: extracting viral RNA from a sample as a template, or reverse-transcription of the RNA as a template, or constructing a plasmid containing a fragment of HRM as a template;

2) using the extracted RNA as a template, performing RT-PCR reaction with the primer pairs P1 and P2 described above; or using the cDNA or plasmid described in the step 1) as a template, using the primer pairs P1 and P2 described above. Carry out a PCR reaction;

3) Perform HRM analysis on the amplified product with reference to the positive control;

If the melting temperature of the sample is consistent with the DV4 positive control, it is determined to be DV4;

If the melting temperature of the sample is consistent with the DV2 positive control, it is determined as DV2;

If the melting temperature of the sample is consistent with the DV3 positive control, it is determined to be DV3;

If the melting temperature of the sample is consistent with the positive control of the DV1 or DV4 standard strain, it is determined to be the DV1 or DV4 standard strain;

4) Distinguish between DV1 and DV4 standard strains:

Repeat the operation of step 2) except that the reference P1 and P2 are replaced with the primer pairs P1 and P3 described above, and other operations are unchanged; and the amplified product is subjected to HRM analysis with reference to the positive control;

If the melting temperature of the sample is consistent with the DV1 positive control, it is determined to be DV1;

If the melting temperature of the sample is identical to the DV4 standard strain positive control, it is judged to be the DV4 standard strain.

Further, the reaction system of the RT-PCR described in the step 2) is:

RNA template .......................................... 2μl

10 μmol of primer P1.......................................... 0.4μl

10μmol primer P2 or P3 ...............................0.4μl

TaKaRa Ex Taq HS ...............................0.4μl

PrimeScript RT enzyme Mix II ...............0.4μl

2X One Step RT-PCR Buffer III ..............10μl

LC green dye ...................................0.5μl

RNase Free ddH ₂ O .......................... Add up to 20μl.

Further, the reaction system of the PCR described in the step 2) is:

Premix Taq ^TM ........................................25μl

Plasmid template ........................................... 2μl

10μmol upstream primer P1 ................................0.5μl

10μmol downstream primer P2/P3 ..........................0.5μl

LC green dye ........................................0.5μl

ddH ₂ O ............................................... .... to 50μl.

Further, the reaction procedure of the RT-PCR described in the step 2) is: 42 ° C 30 min; pre-denaturation at 94 ° C for 2 min; denaturation at 94 ° C for 30 s, annealing at 53 ° C for 30 s, extension at 72 ° C for 30 s, 35 cycles; 72 ° C for 8 min.

Further, the PCR reaction procedure is: pre-denaturation at 94 ° C for 5 min; denaturation at 94 ° C for 30 s, annealing at 53-55 ° C for 30 s, extension at 72 ° C for 30 s; 35 cycles; and 72 ° C for 8 min.

The beneficial effects of the invention are:

1) The present invention establishes for the first time an RT-PCR-HRM or PCR-HRM primer and method for rapidly distinguishing different serotypes of dengue virus, and the four serotypes of dengue virus can be easily identified by the primers and methods of the present invention.

2) The operation of the invention is simple: only the fluorescent saturated dye can be added before the RT-PCR or PCR-HRM reaction; the detection speed is fast and the high throughput: the whole operation process only takes 2 to 3 hours, and the virus cell culture is not required. Greatly shortens the time required for typing; low cost, no specific probe is required, and the cost of saturated dye per sample is 1 RMB; high accuracy, good specificity, good repeatability, accurate, fast and high-throughput analysis, which is conducive to popularization and application in clinical practice.

3) The RT-PCR-HRM or PCR-HRM primers of the present invention have good versatility, and have good amplifying properties for the four serotypes of dengue viruses, which contribute to the improvement of PCR efficiency and the reduction of virus identification typing. time.

4) The RT-PCR-HRM or PCR-HRM primer of the present invention has good specificity, except that it can bind to four serotypes of dengue virus, does not bind to other common flavivirus-like viral RNA, and specifically amplifies dengue virus. RNA facilitates the improvement of the serotype analysis of the present invention.

DRAWINGS

Figure 1 is an electrophoresis pattern of a 67 bp RT-PCR product of four serotype DV cell culture supernatants;

Figure 2 is a graph showing the normalized melting curve of a 67 bp RT-PCR product of four serotype DV cell culture supernatants;

Figure 3 is a graph showing the peak melting curve of a 67 bp RT-PCR product of four serotype DV cell culture supernatants;

Figure 4 is a 67 bp of a plasmid containing the gene of interest in the DV1, DV2, DV3, DV4 and DV4 standard strains, respectively. Electropherogram of the PCR product;

Figure 5 is a 67 bp plasmid containing the gene of interest in the DV1, DV2, DV3, DV4 and DV4 standard strains, respectively. Standardized melting curve of PCR products;

Figure 6 is a 67 bp of a plasmid containing the gene of interest in the DV1, DV2, DV3, DV4 and DV4 standard strains, respectively. Peaking melting curve of PCR product;

Figure 7 is an electrophoresis pattern of a 45 bp PCR product of a plasmid containing the gene of interest in the DV1 and DV4 standard strains, respectively;

Figure 8 is a graph showing the normalized melting curve of a 45 bp PCR product of a plasmid containing the gene of interest in the DV1 and DV4 standard strains, respectively;

Figure 9 is a graph showing the peaking melting curve of a 45 bp PCR product of a plasmid containing the gene of interest in the DV1 and DV4 standard strains, respectively;

Figure 10 is a 67 bp typing sequence in plasmid sequencing results of a 280 bp gene of interest in DV1;

Figure 11 is a 67 bp typing sequence in plasmid sequencing results of a 280 bp gene of interest in DV2;

Figure 12 is a 67 bp typing sequence in plasmid sequencing results of a 280 bp gene of interest in DV3;

Figure 13 is a 67 bp typing sequence in plasmid sequencing results of a 280 bp gene of interest in DV4;

Figure 14 is a 67 bp typing sequence in the 280 bp plasmid sequencing result of the DV4 standard strain of Liffei biosynthesis.

detailed description

The present invention will be further described below in conjunction with specific embodiments, but is not limited thereto.

Example 1: Rapidly distinguishing base sites and gene fragments of different serotypes of dengue virus

(1) The present invention has been subjected to extensive experimental studies and found that the following base sites can be used to rapidly distinguish different serotypes of dengue virus: the 10648th base G in the genome of the DV1 standard strain of Genbank number EU848545.1, the 10671th base Base T, base 10672 base A, base 10675 base G;

Genbank number is AF038403.1 The DV2 standard strain genome has the 10638th base A, the 10661th base C, the 10662th base T, the 10665th base G;

The Genbank number is the 106th base G of the DV3 standard strain genome of M93130.1, the 10633th base C, the 10634th base T, the 10637th base G;

Genbank number is the 10570th base A in the genome of the DV4 standard strain of AY947539.1, the base T of the 10593th base, the base G of the 10594th base, and the base A of the 10597th.

(2) After further experimental research, the present invention found that the following gene fragments can be used to rapidly distinguish different serotypes of dengue virus:

67bp gene fragment:

5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCTACAGCATCATTCCAGGCACAG-3' (SEQ ID NO: 171);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’ (SEQ ID NO: 172);

5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’ (SEQ ID NO: 173);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTACAACATCAATCCAGGCACAG-3’ (SEQ ID NO: 174);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAG-3’ (SEQ ID NO: 175);

45bp gene fragment:

5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 176);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 177);

5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 178);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 179);

5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 180).

Example 2 RT-PCR-HRM or PCR-HRM primers for rapid differentiation of four serotypes of dengue virus

After the experimental screening of a large number of primers designed, the present invention finds an RT-PCR-HRM or PCR-HRM primer which can be used to rapidly distinguish different serotypes of dengue virus, and the target fragment amplified by the primer contains the following base. Base point:

The Genbank number is the 10648th base G in the genome of the DV1 standard strain of EU848545.1, the 10671th base T, the 10672th base A, the 10675th base G;

Genbank number is AF038403.1 The DV2 standard strain genome has the 10638th base A, the 10661th base C, the 10662th base T, the 10665th base G;

The Genbank number is the 106th base G of the DV3 standard strain genome of M93130.1, the 10633th base C, the 10634th base T, the 10637th base G;

Genbank number is the 10570th base A in the genome of the DV4 standard strain of AY947539.1, the base T of the 10593th base, the base G of the 10594th base, and the base A of the 10597th.

Further screening by experiments revealed that the combination of primers P1, P2 and P3 was the best for distinguishing four serotypes of dengue virus by RT-PCR-HRM method.

Wherein the base sequence of the primer P1 is selected from at least one of SEQ ID NOS: 1 to 68.

The base sequence of the primer P2 is selected from at least one of SEQ ID NOS: 69 to 96.

The base sequence of the primer P3 is selected from at least one of SEQ ID NOS: 97 to 170.

Example 3: A method for rapidly distinguishing RT-PCR-HRM of four serotypes of dengue virus

1) Extraction of RNA from DV:

The clinically isolated dengue virus cell culture supernatant was provided by the Eighth People's Hospital of Guangzhou. Using the Tiangen Virus DNA/RNA Extraction Kit (OSR-M202) in TGuide Dengue virus RNA in the supernatant of four clinically isolated serotypes of dengue virus cell culture fluid was separately extracted from the M16 automatic nucleic acid extractor as a template for subsequent one-step RT-PCR.

2) One-step RT-PCR:

Using ^{Takara One Step PrimeScript TM RT-PCR} Kit (Perfect Real Time) Kit, containing TaKaRa Ex Taq HS, PrimeScript RT enzyme Mix II, 2X One Step RT-PCR Buffer III and the like. 20 μl of the system: The amount of the RNA template added to 5 μl, based on the actual concentration, is generally 2 μl. 0.4 μl of TaKaRa Ex Taq HS was added, 0.4 μl of each of the upstream and downstream primers, 0.4 μl of PrimeScript RT enzyme Mix II, 10 μl of 2X One Step RT-PCR Buffer III, and 0.5 μl of LC green dye. Supplement to 20 μl with RNase Free ddH ₂ O.

The specific reaction system in this embodiment is:

RNA template ........................................ 2μl

10 μmol primer P1 ......................................0.4μl

10 μmol primer P2 ......................................0.4μl

TaKaRa Ex Taq HS .............................0.4μl

PrimeScript RT enzyme Mix II .............0.4μl

2X One Step RT-PCR Buffer III ...........10μl

LC green dye ................................0.5μl

RNase Free ddH ₂ O ....................... Add up to 20μl.

The sequence of the above primer P1 is 5'-AAACAGCATATTGACGCTGG-3' (SEQ ID) NO: 3);

The sequence of primer P2 was 5'-CTGTGCCTGGAATGATG -3' (SEQ ID NO: 69).

The RT-PCR reaction procedure is: 42 ° C 30 min; pre-denaturation at 94 ° C for 2 min; denaturation at 94 ° C for 30 s, annealing at 53 ° C for 30 s, extension at 72 ° C for 30 s, cycle 35 times; final extension at 72 ° C for 8 min, amplification product in Rotor-Gene Analyze on the Q instrument.

3) Analysis of results:

Electrophoretic detection: The electropherogram of the above RT-PCR product is shown in Fig. 1. As can be seen from Fig. 1, the four serotypes of dengue virus obtained in the present embodiment can be amplified with a 67 bp gene fragment, and Sequencing (completed by Shanghai Shenggong Bioengineering Co., Ltd.), sequencing results showed that DV1 (dengue virus type 1), DV2 (dengue virus type 2), DV3 (dengue virus type 3) 67bp fragments and corresponding The standard strain is identical, and the 67 bp fragment of DV4 (dengue virus type 4) used in this example has a base mutation with the DV4 standard strain (the 47th base of the 67 bp fragment is mutated to A), ie, SEQ ID NO: 174 has a base mutation with the 47th base of SEQ ID NO: 175.

HRM analysis:

The above RT-PCR products are all in Rotor-Gene The HRM (High Resolution Melting Curve Analysis Technology) analysis was performed on the Q instrument, and the analysis results are shown in Fig. 2 and Fig. 3.

Figure 2 shows the supernatant of four serotype DV cell culture supernatants 67 bp RT- The normalized melting curve of the PCR product; it can be seen that the melting curves of DV1, DV2, DV3 and DV4 are separated from each other, indicating that the designed primers P1 and P2 are suitable for HRM analysis of the four serotypes DV, and can distinguish four serotypes. Dengue virus.

Figure 3 shows the supernatant of four serotype DV cell culture supernatants The peak melting curve of RT-PCR products; it can be seen that the melting temperatures (Tm) of DV1, DV2, DV3 and DV4 are different, the melting temperature of DV4 is 76.9 °C, and the melting temperatures of DV1, DV2 and DV3 are 77.6 ° C, 77.9 ° C and 78.16 ° C.

Example 4: A method for rapidly distinguishing PCR-HRM of four serotypes of dengue virus

In order to further analyze whether the primers P1 and P2 can distinguish the DV1, DV2, DV3, DV4, and DV4 standard strains, this example constructs a 280 bp plasmid containing 67 bp and 45 bp fragmentation fragments, and additionally adds a synthetic gene fragment. A plasmid sample (a 280 bp fragment of the DV4 standard strain, comprising a 67 bp and 45 bp fragment, was cloned into the puc57 plasmid and synthesized by Shanghai Lifei Biotechnology Co., Ltd.).

1) Preparation of a plasmid sample containing the DV 280 bp gene of interest:

Upstream primer 5'-GCWGCCTGTAGCTCC-3' (SEQ ID) NO: 181), a downstream primer 5'-CTGTGCCTGGAATGATG-3' (SEQ ID NO: 182) amplifying a 280 bp fragment of the dengue virus gene (containing 67 bp and 45 bp) HRM typing fragment), purified and recovered PCR product, and ligated with Takara pMD-18T vector.

Vector ligation reaction system (10 μl):

Ligation Solution I	5μl
Purified dengue virus 280 bp DNA product	4.5μl
pMD-18T vector	0.5μl
total capacity	10μl

Reaction conditions: 16 ° C, 4 h or more.

After the compatibilized cells frozen at -70 °C were thawed on ice, 5 μl of the ligation product was added, and the mixture was gently mixed and ice-cooled for 30 min. The 42 ° C water bath was heat shocked for 90 sec and then quickly placed back on ice for 2 min. Add 400μl The LB liquid medium was cultured at 37 ° C at 200 r / min - 220 r / min for 45 min to restore the resistance of the plasmid. Centrifuge at 4 °C 4000r/min for 5 min, discard the supernatant 400 μl, mix the remaining 100 μl of the bacterial solution, and apply the ampicillin plate, incubate at 37 ° C for 30 min (plate is placed), until the bacterial solution is completely absorbed, and then invert the plate for about 12 h. ~16h, to a single colony. A single colony was picked up in 5 ml of ampicillin-containing LB liquid medium with a sterile toothpick, and cultured at 37 ° C for 200 h/min-220 r/min for 12 h to 16 h, and identified by PCR.

2) Extraction of plasmid:

Extract DV1, DV2, DV3, DV4 with TIANprep Mini Plasmid Kit Plasmid Kit , plasmid of DV4 standard strain.

3) PCR amplification:

The PCR amplification system of the DV1, DV2, DV3, DV4, and DV4 standard strain plasmids is:

Premix Taq ^TM (TaKaRa Taq ^TM Version 2.0) ...........25μl

Plasmid template .................................................. ................2μl

10μmol upstream primer P1 ...........................................0.5 Ll

10 μmol downstream primer P2 ...........................................0.5 Ll

LC green dye .................................................. ........0.5μl

ddH ₂ O ............................................... ...................... add up to 50μl.

The sequence of the above primer P1 is 5'-AAACAGCATATTGACGCTGG-3' (SEQ ID) NO: 3);

The sequence of primer P2 was 5'-CTGTGCCTGGAATGATG -3' (SEQ ID NO: 69).

PCR reaction procedure:

Pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s (downstream primer for P2; downstream primer for P3 with annealing temperature of 55 °C), 72 °C for 30 s; cycle 35 times; 72 °C for 8 min.

4) Analysis of results:

Electrophoretic detection: The electrophoresis pattern of the above PCR amplification product is shown in Fig. 4. As can be seen from Fig. 4, all the samples in this example can be amplified with a 67 bp gene fragment. The constructed 280 bp plasmid was sequenced (completed by Shanghai Shenggong Bioengineering Co., Ltd.) and the sequencing results were correct.

HRM analysis: The above PCR products are all in Rotor-Gene The HRM (High Resolution Melting Curve Analysis Technology) analysis was performed on the Q instrument, and the analysis results are shown in Fig. 5 and Fig. 6.

Figure 5 is a 67 bp of four serotype DV plasmids and a synthetic DV4 standard strain plasmid. The normalized melting curve of the PCR product can be seen; the normalized melting curves of DV2, DV3, and DV4 can be well distinguished, but the curves of the DV1 and DV4 standard strains are mixed together and cannot be effectively distinguished.

Figure 6 shows the 67 bp of four serotype DV plasmids and the synthetic DV4 standard strain plasmid. The peaking melting curve of the PCR product; similar to Figure 5, the peaking melting curves of DV2, DV3, and DV4 can be well distinguished. The melting temperature of DV4 is 79.29±0.028°C, and the melting temperature of DV2 is 80.52±0.027°C. The melting temperature of DV3 is 80.93±0.025°C, and the melting temperature of DV1 or DV4 standard strain is 80.26±0.038°C, but the curves of DV1 and DV4 standard strains are mixed together and cannot be effectively distinguished.

5) Classification of DV1 and DV4 standard strains:

The plasmids of the DV1 and DV4 standard strains were separately subjected to PCR amplification, except that the primers used were P1 and P3, and the other operations were the same as the above step 2);

The sequence of the above primer P1 is 5'-AAACAGCATATTGACGCTGG-3' (SEQ ID) NO: 3);

The sequence of primer P3 is 5'-GAGACAGCAGGATCTCTG-3' (SEQ ID NO: 101);

Electrophoretic detection: The electrophoresis pattern of the above PCR amplification products is shown in Fig. 7. As can be seen from Fig. 7, both the DV1 and DV4 standard strain samples can be amplified with a 45 bp gene fragment. The amplified gene fragments were sequenced separately (completed by Shanghai Shenggong Bioengineering Co., Ltd.), and the sequencing results were correct.

HRM analysis: The above PCR products are all in Rotor-Gene The HRM (High Resolution Melting Curve Analysis Technology) analysis was performed on the Q instrument, and the analysis results are shown in Fig. 8 and Fig. 9.

Figure 8 is a 45 bp of the plasmid of DV1 and DV4 standard strains. The PCR product was normalized to the melting curve; it can be seen that the melting curves of the DV1 and DV4 standard strains are separated from each other, indicating that the designed primers P1 and P3 are suitable for HRM analysis of the two serotype DVs (DV1 and DV4).

Figure 9 is a 45 bp plasmid of the DV1 and DV4 standard strains. The peak product melting curve of the PCR product; it can be seen that the melting temperature (Tm) of the DV1 and DV4 standard strains is different, the melting temperature of the DV4 standard strain is 76.13±0.023°C, and the melting temperature of DV1 is 76.77±0.031°C, which can be Very well separated.

Figure 10 is a sequencing sequence of the corresponding 67 bp base in the 280 bp base fragment sequencing result in DV1;

Figure 11 is a sequencing sequence of the corresponding 67 bp base in the 280 bp base fragment sequencing result in DV2 (sequence map is a reverse complement sequence);

Figure 12 is a sequencing sequence of the corresponding 67 bp base in the 280 bp base fragment sequencing result in DV3;

Figure 13 is a sequencing sequence of the corresponding 67 bp base in the 280 bp base fragment sequencing result in DV4;

Figure 14 is a sequencing sequence of the corresponding 67 bp base in the 280 bp base fragment of the DV4 standard strain of the Lifei biosynthesis.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and combinations thereof may be made without departing from the spirit and scope of the invention. Simplifications should all be equivalent replacements and are included in the scope of the present invention.

Claims (12)

1.  The use of base sites for rapid differentiation of different serotypes of dengue virus, the base sites are:

   The Genbank number is the 10648th base G in the genome of the DV1 standard strain of EU848545.1, the 10671th base T, the 10672th base A, the 10675th base G;

   The Genbank number is AF038403.1 in the genome of the DV2 standard strain, the 10638th base A, the 10661th base C, the 10662th base T, the 10665th base G;

   The Genbank number is the 106th base G of the DV3 standard strain genome of M93130.1, the 10633th base C, the 10634th base T, the 10637th base G;

   Genbank number is the 10570th base A in the genome of the DV4 standard strain of AY947539.1, the base T of the 10593th base, the base G of the 10594th base, and the base A of the 10597th.
2. The use of gene fragments to rapidly distinguish between different serotypes of dengue viruses, which are:

   67bp gene fragment:

   5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCTACAGCATCATTCCAGGCACAG-3' (SEQ ID NO: 171);

   5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’ (SEQ ID NO: 172);

   5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’ (SEQ ID NO: 173);

   5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTACAACATCAATCCAGGCACAG-3’ (SEQ ID NO: 174);

   5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAG-3’ (SEQ ID NO: 175);

   45bp gene fragment:

   5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 176);

   5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 177);

   5'-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 178);

   5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 179);

   5'-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3' (SEQ ID NO: 180).
3. An RT-PCR-HRM or PCR-HRM primer for rapidly distinguishing different serotypes of dengue virus, the target gene fragment amplified by the primer contains the following base sites:

   The Genbank number is the 10648th base G in the genome of the DV1 standard strain of EU848545.1, the 10671th base T, the 10672th base A, the 10675th base G;

   The Genbank number is AF038403.1 in the genome of the DV2 standard strain, the 10638th base A, the 10661th base C, the 10662th base T, the 10665th base G;

   The Genbank number is the 106th base G of the DV3 standard strain genome of M93130.1, the 10633th base C, the 10634th base T, the 10637th base G;

   Genbank number is the 10570th base A in the genome of the DV4 standard strain of AY947539.1, the base T of the 10593th base, the base G of the 10594th base, and the base A of the 10597th.
4. A RT-PCR-HRM or PCR-HRM primer for rapidly distinguishing different serotypes of dengue virus according to claim 3, said primers comprising primers P1, P2 and P3;

   The base sequence of the primer P1 is selected from at least one of SEQ ID NOS: 1-68;

   The base sequence of the primer P2 is selected from at least one of SEQ ID NOS: 69 to 96;

   The base sequence of the primer P3 is selected from at least one of SEQ ID NOS: 97 to 170.
5. A RT-PCR-HRM or PCR-HRM primer for rapidly distinguishing different serotypes of dengue virus according to claim 4, said primers comprising primers P1, P2 and P3;

   The base sequence of the primer P1 is selected from at least one of SEQ ID NOS: 1 to 20;

   The base sequence of the primer P2 is selected from at least one of SEQ ID NOS: 69 to 80;

   The base sequence of the primer P3 is selected from at least one of SEQ ID NOS: 97 to 120.
6. A RT-PCR-HRM or PCR-HRM primer for rapidly distinguishing different serotypes of dengue virus according to claim 5, said primers comprising primers P1, P2 and P3;

   The base sequence of the primer P1 is selected from at least one of SEQ ID NOS: 1 to 4;

   The base sequence of the primer P2 is selected from at least one of SEQ ID NOS: 69 to 71;

   The base sequence of the primer P3 is selected from at least one of SEQ ID NOS: 99 to 101.
7. An RT-PCR-HRM or PCR-HRM reagent for rapidly distinguishing different serotypes of dengue virus, characterized in that the reagent comprises the primers P1, P2 and P3 according to any one of claims 3 to 6.
8. An RT-PCR-HRM or PCR-HRM method for rapidly distinguishing different serotypes of dengue virus, comprising the steps of:

   1) Preparation of a template: extracting viral RNA from a sample as a template, or reverse-transcription of the RNA as a template, or constructing a plasmid containing a fragment of HRM as a template;

   2) using the extracted RNA as a template, performing RT-PCR reaction on the primer pair P1 and P2 according to any one of claims 3 to 6; or using the cDNA or plasmid described in the step 1) as a template, and claim 3 The primers of any of the groups 6 to 6 perform PCR reaction on P1 and P2;

   3) Perform HRM analysis on the amplified product with reference to the positive control;

   If the melting temperature of the sample is consistent with the DV4 positive control, it is determined to be DV4;

   If the melting temperature of the sample is consistent with the DV2 positive control, it is determined as DV2;

   If the melting temperature of the sample is consistent with the DV3 positive control, it is determined to be DV3;

   If the melting temperature of the sample is consistent with the positive control of the DV1 or DV4 standard strain, it is determined to be the DV1 or DV4 standard strain;

   4) Distinguish between DV1 and DV4 standard strains:

   Repeat the operation of step 2) except that the reference P1 and P2 are replaced with the primer pairs P1 and P3 according to any one of claims 3 to 6, and the other operations are unchanged; the amplification product is carried out with reference to the positive control. HRM analysis;

   If the melting temperature of the sample is consistent with the DV1 positive control, it is determined to be DV1;

   If the melting temperature of the sample is identical to the DV4 standard strain positive control, it is judged to be the DV4 standard strain.
9. The method according to claim 8, wherein the reaction system of the RT-PCR in step 2) is:

   RNA template .................................................. ...2μl

   10 μmol primer P1 ......................................0.4μl

   10μmol primer P2 or P3 ....................................0.4μl

   TaKaRa Ex Taq HS ....................................0.4μl

   PrimeScript RT enzyme Mix II ....................0.4μl

   2X One Step RT-PCR Buffer III ..................10μl

   LC green dye .......................................0.5μl

   RNase Free ddH ₂ O .............................. Add up to 20μl.
10. The method according to claim 8, wherein the reaction system of the PCR in step 2) is:

    Premix Taq ^TM .......................................25μl

    Plasmid template .................................................. ....2μl

    10μmol upstream primer P1 .....................................0.5μl

    10μmol downstream primer P2/P3 ................................0.5μl

    LC green dye .........................................0.5μl

    ddH ₂ O ............................................... ........... add up to 50μl.
11. The method according to claim 8 or 9, wherein the reaction procedure of the RT-PCR in the step 2) is: 42 ° C for 30 min; 94 ° C pre-denaturation for 2 min; 94 ° C denaturation for 30 s, 53 ° C annealing for 30 s, 72 °C extended for 30s, cycled 35 times; 72 °C extended for 8min.
12. The method according to claim 9 or 10, wherein the PCR reaction procedure is: pre-denaturation at 94 ° C for 5 min; denaturation at 94 ° C for 30 s, annealing at 53-55 ° C for 30 s, extension at 72 ° C for 30 s; cycle 35 times; The final extension of °C is 8 min.