Composition, kit and method for detecting and typing three mosquito-borne arthroviruses and application

Abstract

The invention belongs to the field of molecular biological detection, and particularly relates to combined nucleic acid detection of three typical mosquito-borne arthroviruses, namely, dengue virus, Zika virus and Chikungunya virus, and four serotypes of the dengue virus are typed by using a melting curve matched molecular beacon design method. The method specifically comprises the following steps: distinguishing and detecting three typical mosquito-borne arbo viruses by using an amplification curve, and marking pathogens in each tube on different probes by using different fluorophores so as to distinguish amplification signals. A melting curve is used for distinguishing four different types of viruses of the dengue virus, and Tm value characteristic peak signals are distinguished by combining a molecular beacon and a locked nucleic acid probe aiming at each type of specific target. According to the invention, clinical differential diagnosis of the three viruses is facilitated, and an auxiliary means is provided for guiding medication for different clinical manifestations in dengue virus typing detection.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology detection, and in particular to the combined nucleic acid detection of three typical mosquito-borne arboviruses: dengue virus, Zika virus and chikungunya virus, and simultaneously typing the four serotypes of the dengue virus by using a melting curve combined with a molecular beacon design method. Background Art

[0002] Mosquitoes are one of the most harmful vectors. More than 3,500 mosquito species have been reported worldwide, of which more than 300 have been discovered and identified in my country, accounting for about one-tenth of all mosquito species. Mosquitoes can carry and transmit a wide variety of pathogens, and cause a variety of serious mosquito-borne infectious diseases, such as dengue fever, chikungunya fever and Zika virus disease. In recent years, with global warming, the temperature has generally risen, the probability of mosquito-borne diseases has greatly increased, and the geographical distribution of diseases has become more extensive. According to WHO estimates, there are 96 million new cases of dengue fever, 693,000 cases of chikungunya fever, and 500,000 cases of Zika virus disease worldwide each year, and the disease burden and harm are extremely serious. In China, there are many types of mosquitoes and a wide range of activities. my country has a large geographical span and rich local pathogens and vectors. In addition, with the growth of my country's population, the expansion of urbanization, frequent international trade, and the increase in international tourism, the number of imported mosquito-borne diseases has increased, and the public health problems they bring have received more and more attention.

[0003] Given the widespread spread of arboviruses and the unpredictability of outbreaks, many arboviruses cause serious human diseases, and the symptoms caused by arboviruses are not obviously specific, making it difficult to make differential diagnosis. Improving the detection capabilities of arboviruses is particularly important. Dengue fever, chikungunya fever, and Zika virus disease are mosquito-borne diseases that have a significant impact worldwide and have a certain range of spread in my country. They all have relatively similar epidemiological and clinical characteristics, and the main transmission vectors are Aedes aegypti and Aedes albopictus. It is necessary to make differential diagnosis of the three viruses in clinical practice.

[0004] Dengue virus belongs to the genus Flaviviridae. Dengue virus particles are spherical with a diameter of 45-55nm. Dengue fever caused by dengue virus causes fever, flushing of the skin, leukopenia and thrombocytopenia syndrome. Dengue virus not only causes dengue fever, but also causes severe symptoms such as dengue hemorrhagic fever and dengue shock syndrome, as well as other unusual symptoms. Dengue fever has evolved from a sporadic disease to a major public health problem, with significant social and economic impacts due to the expansion of geographical scope, number of cases and severity of the disease. There are four serotypes of dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4), all of which can infect humans. Among them, the severity rate and mortality rate of type 2 are higher than those of other types. In recent years, with the deepening of research, it has been found that the infection rate of DENV 1 / 3 / 4 types is increasing year by year, and the clinical symptoms after infection with different types are different. Therefore, it is necessary to use dengue virus typing detection to guide medication for different clinical manifestations. Summary of the invention

[0005] In view of this, the present invention, in a first aspect, provides a composition for detecting and typing three mosquito-borne arboviruses, comprising:

[0006] The first group used for fluorescent probe method:

[0007] A primer pair and a probe for detecting dengue virus as shown in SEQ ID NOs: 1 to 3;

[0008] Primer pairs and probes for detecting Zika virus as shown in SEQ ID NOs: 7 to 9;

[0009] A primer pair and a probe for detecting Chikungunya virus as shown in SEQ ID NOs: 10 to 12; and

[0010] The second set for melting curve method:

[0011] A primer pair and a probe for detecting dengue virus type I as shown in SEQ ID NOs: 13 to 15;

[0012] A primer pair and a probe for detecting dengue virus type II as shown in SEQ ID NOs: 16 to 18;

[0013] A primer pair and a probe for detecting dengue virus type III as shown in SEQ ID NOs: 19 to 21;

[0014] The primer pair and probe for detecting dengue virus type IV are shown in SEQ ID NOs: 22-24.

[0015] Furthermore, the composition also includes an internal standard primer pair and a probe as shown in SEQ ID NOs: 4 to 6.

[0016] The composition provided by the present invention first uses a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and identification of three typical mosquito-borne arboviruses: dengue virus, Zika virus and chikungunya virus in a single-tube reaction system. At the same time, the four serotypes (types I to IV) of dengue virus are typed by using a melting curve combined with a molecular beacon probe design method. The composition of the present invention can still accurately detect samples as low as 600 copies / mL in each channel.

[0017] Multiplex nucleic acid testing is widely used in screening risk factors, pathogen typing, and detecting susceptibility to genetic diseases. Multiplex testing is not only conducive to timely, cost-effective testing, but also provides more sample information compared to single-plex testing. The common multiplex nucleic acid testing method is to design primer probes that match the highly conserved sequences of different pathogens in the polymerase chain reaction (PCR) system to achieve the detection of multiple pathogens in the same tube. Since the number of detection channels, fluorophores, and targets is equal in the classic multiplex real-time fluorescence quantitative PCR, multiplex detection can be performed in one reaction system by simultaneously detecting different fluorophore-labeled probes. For example, a real-time thermal cycler equipped with four detection channels can perform up to four-plex detection simultaneously. Using melting temperature (Tm) as the second dimension of multiplexing, higher multiplex detection can be achieved on the basis of real-time PCR. After the amplification reaction is completed, the melting curve is generated by gradually increasing the temperature while monitoring the fluorescence signal at each step. There is a characteristic peak (Tm value) on the melting curve, which is proportional to the GC content. This characteristic peak can distinguish specific products from other products (such as primers). In a multiplex PCR reaction system, probes with different GC contents are designed for different targets, so that the melting curves of different targets in the same detection channel have different peaks, thus achieving the effect of simultaneously detecting multiple targets with a single fluorescence channel.

[0018] Fluorescence quantitative PCR (polymerase chain reaction), in this reaction system, contains a pair of specific primers and a TaqMan probe, which is a specific oligonucleotide sequence, with a reporter fluorescent group and a quencher fluorescent group labeled at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; if there is a target sequence in the reaction system, the probe binds to the template during the PCR reaction, and the DNA polymerase uses the exonuclease activity of the enzyme to enzymatically degrade the probe along the template, and the reporter group is separated from the quencher group, emitting fluorescence. For each amplified DNA chain, a fluorescent molecule will be generated. The fluorescence quantitative PCR instrument can monitor the number of cycles (Ct value) at which the fluorescence reaches the preset threshold value, which is related to the concentration of pathogen nucleic acid. The higher the concentration of pathogen nucleic acid, the smaller the Ct value. In the end, the infection can be judged based on the Ct value.

[0019] Molecular beacons are double-labeled single-stranded oligonucleotide probes with a hairpin structure consisting of a loop and a neck. The neck is composed of complementary sequences with high C / G content at both ends and no homology to the target gene; the loop is composed of a complementary sequence of the target sequence. In a reaction system without the presence of a target gene, the complementary sequences at both ends combine to form a hairpin structure during annealing; in a system containing a target sequence, the loop hybridizes with the target sequence due to intermolecular forces during annealing, the molecular beacon is elongated, the spatial distance between the fluorescent group and the quenching group is elongated, the fluorescence quenching efficiency is reduced, and a change in the fluorescence signal occurs. Since the neck of the molecular beacon does not participate in hybridization at all, there is a dynamic competition between the complementary hybridization trend of the neck sequence itself and the hybridization between the loop and the target gene, so it has stronger specificity than conventional TaqMan probes.

[0020] In some embodiments, the composition of the present invention may include one or more sets of the above primer pairs and probes. In this article, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0021] Furthermore, the composition of the present invention can be arbitrarily combined into any combination form for detecting corresponding targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe sets corresponding to the targets. These combinations are all included in the present invention.

[0022] Furthermore, the fluorescent groups of the probes in the first group in the composition are different from each other and do not interfere with each other.

[0023] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the first group of the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, FAM, HEX, ROX and CY5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0024] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0025] In some specific embodiments, the probes in the compositions of the present invention are labeled with a fluorescent reporter group at the 5' end.

[0026] Furthermore, the fluorescent reporter group of the probe for detecting dengue virus as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting Zika virus as shown in SEQ ID NO:9 is ROX; and the fluorescent reporter group of the probe for detecting chikungunya virus as shown in SEQ ID NO:12 is CY5.

[0027] Furthermore, the fluorescent reporter group of the probe for detecting dengue virus type I as shown in SEQ ID NO:15 is FAM; the fluorescent reporter group of the probe for detecting dengue virus type II as shown in SEQ ID NO:18 is FAM; the fluorescent reporter group of the probe for detecting dengue virus type III as shown in SEQ ID NO:21 is FAM; the fluorescent reporter group of the probe for detecting dengue virus type IV as shown in SEQ ID NO:24 is FAM.

[0028] In some specific embodiments, the 3' end of the probe further has a fluorescence quenching group, such as MGB, BHQ1 or BHQ2.

[0029] Furthermore, the components of the composition exist in a mixed form.

[0030] In a second aspect, a use of the composition of the present invention in preparing a reagent or a kit for detecting and typing three mosquito-borne arboviruses is provided, wherein the three mosquito-borne arboviruses are dengue virus, Zika virus and chikungunya virus.

[0031] In a third aspect, a kit for detecting and typing three mosquito-borne arboviruses is provided, the kit comprising the composition of the present invention.

[0032] Furthermore, in each reaction system, the amount of primer used is 0.05 to 5 μM, and the amount of probe used is 0.05 to 5 μM.

[0033] Furthermore, the concentration range of the probe for detecting dengue virus type I as shown in SEQ ID NO:15 is 0.16-0.4 μM, the concentration range of the probe for detecting dengue virus type II as shown in SEQ ID NO:18 is 0.24-0.56 μM, the concentration range of the probe for detecting dengue virus type III as shown in SEQID NO:21 is 0.08-0.4 μM, and the concentration range of the probe for detecting dengue virus type IV as shown in SEQ ID NO:24 is 0.24-0.64 μM.

[0034] Furthermore, the concentration range of the probe for detecting dengue virus type I as shown in SEQ ID NO:15 is 0.24 μM, the concentration range of the probe for detecting dengue virus type II as shown in SEQ ID NO:18 is 0.32 μM, the concentration range of the probe for detecting dengue virus type III as shown in SEQ ID NO:21 is 0.16 μM, and the concentration range of the probe for detecting dengue virus type IV as shown in SEQ ID NO:24 is 0.32 μM.

[0035] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0036] In a specific embodiment, the negative control is sterile saline, but is not limited to sterile saline, and can also be DEPC H2O.

[0037] In a specific embodiment, the positive control is at least one of a fragment plasmid or a fragment DNA of dengue virus, Zika virus, chikungunya virus, dengue virus type I, dengue virus type II, dengue virus type III, and dengue virus type IV.

[0038] Furthermore, the kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, a reverse transcriptase, a DNA polymerase, dNTPs (T) and Mg 2+ At least one of .

[0039] In some specific embodiments, the reverse transcriptase is Neoscript RT enzyme.

[0040] In some specific embodiments, the DNA polymerase is H-Taq enzyme.

[0041] In a specific embodiment, the kit of the present invention comprises PCR buffer, Neoscript RT enzyme, H-Taq enzyme, dNTPs (T), primers, probe and Mg 2+ .

[0042] Common PCR buffers are composed of Tris-HCl, KCl, MgCl2 and other buffer systems. The total volume in a single PCR reaction tube is generally 20 to 200 μL.

[0043] In a fourth aspect, a method for detecting and typing three mosquito-borne arboviruses for non-diagnostic purposes is provided, the method comprising the following steps:

[0044] (1) Extracting or releasing nucleic acid from the sample to be tested;

[0045] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition of the present invention or the kit of the present invention;

[0046] (3) Obtain and analyze the results.

[0047] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0048] Reverse transcription, temperature 50℃, time 30min, 1 cycle; pre-denaturation and enzyme activation, temperature 95℃, time 1min, 1 cycle; denaturation, temperature 95℃, time 15s, annealing, extension and fluorescence collection, temperature 58℃, time 30s, 45 cycles; melting curve, temperature 50-95℃, fluorescence collection throughout the process, 1 cycle.

[0049] In a specific embodiment, the present invention provides a composition for preparing a reagent for detecting and typing three mosquito-borne arboviruses, the use comprising the following steps:

[0050] (1) Extracting or releasing nucleic acid from the sample to be tested;

[0051] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition of the present invention or the kit of the present invention;

[0052] (3) Obtain and analyze the results.

[0053] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0054] Reverse transcription, temperature 50℃, time 30min, 1 cycle; pre-denaturation and enzyme activation, temperature 95℃, time 1min, 1 cycle; denaturation, temperature 95℃, time 15s, annealing, extension and fluorescence collection, temperature 58℃, time 30s, 45 cycles; melting curve, temperature 50-95℃, fluorescence collection throughout the process, 1 cycle.

[0055] In this article, the term "non-diagnostic purpose" refers to not being intended to obtain information on whether an individual is infected with the above pathogens and related diseases. For example, the presence of the above pathogens can be detected in a test culture.

[0056] In the present invention, the term "primer pair" refers to two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target gene, and the other primer is complementary to another DNA template strand at the other end of the target gene. Alternatively, it refers to two oligonucleotide chains used to specifically amplify the target DNA fragment in the polymerase chain reaction (PCR). This pair of primers corresponds to the two ends of the target DNA sequence, one is complementary to the 5' end of the template strand, called the upstream primer or forward primer; the other is complementary to the 3' end of the template strand, called the downstream primer or reverse primer.

[0057] In the present invention, the term "probe" refers to a nucleic acid sequence (DNA or RNA) with a detection label, a known sequence, and complementary to the target gene.

[0058] In the present invention, the term "nucleotide sequence" refers to the order of bases in DNA or RNA.

[0059] The above scheme of the present invention has the following beneficial effects:

[0060] The present invention designs a tube of reaction solution, and only needs to perform one nucleic acid extraction and one PCR test, so as to simultaneously perform the combined nucleic acid detection of three typical mosquito-borne arboviruses, dengue virus, Zika virus and chikungunya virus, and simultaneously utilize the melting curve in combination with the molecular beacon probe design method to perform the four serotype typing of dengue virus. The above is specifically manifested as: using the amplification curve to distinguish and detect three typical mosquito-borne arboviruses, and using different fluorescent groups to mark different probes for the pathogens in each tube to distinguish the amplification signals. Using the melting curve to distinguish the four different types of dengue virus, combining molecular beacons and locked nucleic acid probes for each type-specific target to distinguish the characteristic peak signal of the Tm value. The present invention is helpful for the differential diagnosis of the above three viruses in clinical practice, and provides an auxiliary means for guiding the use of drugs for different clinical manifestations for the typing detection of dengue virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] 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.

[0062] Figure 1 This is an amplification curve diagram of the joint detection of dengue virus, Zika virus and chikungunya virus by the composition of the embodiment of the present invention;

[0063] Figure 2 This is a melting curve diagram of the joint detection of dengue virus types I to IV by the composition of Example 2 of the present invention;

[0064] Figures 3 to 10 The test results of the composition of the present invention for each virus (dengue virus, internal standard, Zika virus, chikungunya virus, dengue virus type I, dengue virus type II, dengue virus type III, dengue virus type IV) at a detection concentration of 600 copies / mL;

[0065] Fig.11 is a graph showing the specificity detection results of the composition of an embodiment of the present invention;

[0066] Fig.12 It is the amplification curve diagram of Zika virus detected in Example 2 of the present invention and Comparative Example 1;

[0067] Fig.13This is an amplification curve diagram of the combined detection of dengue virus, Zika virus and chikungunya virus by the composition of comparative example 1 of the present invention;

[0068] Fig.14 This is a melting curve diagram of the joint detection of dengue virus types I to IV by the composition of Example 6 of the present invention;

[0069] Fig.15 It is a melting curve diagram of dengue virus types I to IV detected by the composition of comparative example 2 of the present invention. DETAILED DESCRIPTION

[0070] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0071] The disclosed embodiments are described to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

[0072] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0073] Example 1: Primer pairs and probes used in the present invention

[0074] The primer pairs and probes used in the present invention are shown in Table 1 below.

[0075] Table 1

[0076]

[0077]

[0078] Note: "+" specifically refers to LNA modification, for example: "T+C" means locked nucleic acid modification on base "C".

[0079] Among them, the fluorescent reporter group of the probe for detecting dengue virus as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting Zika virus as shown in SEQ ID NO:9 is ROX; the fluorescent reporter group of the probe for detecting Chikungunya virus as shown in SEQ ID NO:12 is CY5; the fluorescent reporter group of the probe for detecting dengue virus type I as shown in SEQ ID NO:15 is FAM; the fluorescent reporter group of the probe for detecting dengue virus type II as shown in SEQ ID NO:18 is FAM; the fluorescent reporter group of the probe for detecting dengue virus type III as shown in SEQ ID NO:21 is FAM; the fluorescent reporter group of the probe for detecting dengue virus type IV as shown in SEQ ID NO:24 is FAM.

[0080] Example 2: Method for detecting and typing three mosquito-borne arboviruses

[0081] (1) Reagent preparation:

[0082] Take the corresponding amount of PCR reaction solution and enzyme mixture (26 μL / person of PCR reaction solution + 4 μL / person of enzyme mixture) in proportion, and mix them thoroughly to form a PCR mixture.

[0083] Preparation of enzyme mixture:

[0084] The enzyme mixture is a mixture of Neoscript RT enzyme and H-Taq enzyme.

[0085] In addition, a negative control using sterile physiological saline as the matrix and a mixed plasmid containing the specific gene target sequences listed in Table 1 were used as the positive control.

[0086] (2) Sample processing and sample addition

[0087] Take 300 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and use conventional nucleic acid extraction or purification reagents according to their instructions to extract nucleic acids.

[0088] 20 μL of the above-treated samples, negative controls and positive controls were pipetted and added to reaction tubes respectively. 30 μL of PCR mixture was added to each reaction tube and the tube cap was covered.

[0089] The real-time fluorescence PCR reaction system is configured according to Table 2 below:

[0090] Table 2

[0091]

[0092]

[0093] (3) PCR amplification was performed on the SLAN-96P fully automatic medical PCR analysis system according to a certain temperature and time setting program. The amplification reaction program is shown in Table 3.

[0094] Table 3

[0095]

[0096] (4) Testing, result analysis and judgment

[0097] Amplification curve

[0098] If the FAM, HEX, CY5 and ROX channels of the amplification curve have obvious S-shaped amplification curves and the Ct value is ≤38, it is judged as positive; if there is no amplification curve (NoCt) or the Ct value is greater than 38 in the FAM, HEX, CY5 and ROX channels, it is judged as negative, as shown in Table 4 below:

[0099] Table 4

[0100]

[0101] Melting curve

[0102] If the amplification curve FAM and HEX channels have obvious S-shaped amplification curves, and the Ct value is ≤38, and the FAM channel of the melting curve detects a characteristic peak corresponding to the Tm range, it indicates that the result is positive for a type of dengue virus from type I to IV; if the HEX channel has an obvious S-shaped amplification curve, but the FAM channel does not have an obvious S-shaped amplification curve, and the melting curve has no characteristic peak or the Tm is not within the range, the result is negative for dengue virus, as shown in Table 5 below:

[0103] Table 5

[0104]

[0105] Example 3: Test results of the test samples of the composition of the present invention

[0106] The primer pair and probe shown in Example 1 were used to verify the sample according to the method of Example 2. The amplification curve of the detection result is shown in FIG. Figure 1 As shown, the results show that dengue virus, Zika virus and chikungunya virus can be jointly detected and distinguished; the melting curve of the detection results is shown in Figure 2 As shown, the results showed that dengue virus type I, dengue virus type II, dengue virus type III and dengue virus type IV could be distinguished.

[0107] Example 4: Sensitivity of the composition of the present invention

[0108] The composition in Example 1 of the present invention was used to perform LOD (sensitivity) detection on each pathogen according to the method in Example 2, and multiple PCR detection was performed on the fluorescent quantitative PCR instrument for the lowest concentration of each target positive plasmid. The results are as follows: Figures 3 to 10 As shown, it is demonstrated that the sensitivity of the composition of the present invention is 600 copies / mL.

[0109] Example 5. Specificity of the composition of the present invention

[0110] Using the fluorescent PCR reaction system in Example 2 of the present invention, according to the detection method in Example 2, specificity testing was performed in reaction systems containing other relevant pathogens such as Japanese encephalitis virus, West Nile virus, mumps virus, norovirus, yellow fever virus, and rubella virus. The test results are as follows: Fig.11 As shown, each target channel has no non-specific amplification and no cross reaction, which shows that the composition provided by the present invention has good specificity.

[0111] Comparative Example 1: Other primers and probes designed by the present invention with poor effects

[0112] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be excluded at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. It is necessary to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection) and consider the mutual interference between different primers and probes, so the primers and probes need to be carefully designed. Therefore, the inventors designed some other primers and probes.

[0113] This comparative example refers to the reaction system and detection method of Example 2, except that the primer pair probe sequence of Zika virus is different (as shown in Table 6 below). In addition to the above differences, the other materials and process operations used in this comparative example are strictly consistent with those of Example 2.

[0114] The primer pair and probe (SEQ ID NO: 7-9) in Example 1 and the primer pair and probe (SEQ ID NO: 25-27) in Comparative Example 1 were used to perform Zika virus single detection, and the detection results are as follows: Fig.12 As shown, from Fig.12 The amplification curve shows that the Zika virus is more effective when detected alone.

[0115] The primer pairs and probes (SEQ ID NOs: 25-27) in Comparative Example 1 were used to replace the primer pairs and probes (SEQ ID NOs: 7-9) in Example 1, and combined with other primer pairs and probes in Example 1 to form a composition (SEQ ID NOs: 1-6, 10-12 and 25-27) for specific gene detection of dengue virus, Zika virus and chikungunya virus. The results are as follows: Fig.13 As shown. Fig.13 It can be seen that after the combined detection of the three pathogens, the Zika virus will have a serious target missed detection. In summary, the results of the two single tests are not much different, but the effect becomes worse after the joint test. This fully demonstrates the superiority of the composition of the present invention, and the mutual interference between different primer pairs and probes is small. In other words, the combination of primers and probes in the present invention is irreplaceable and replaceable.

[0116] Table 6

[0117]

[0118] Example 6. Test results of the test samples of the composition of the present invention

[0119] The primer pairs and probes shown in Example 1 were used to verify the samples according to the method of Example 2, except that: in the real-time fluorescence PCR reaction system, the volume of probe SEQ ID NO: 15 was 0.5 μL (concentration was 0.4 μM); the volume of probe SEQ ID NO: 18 was 0.7 μL (concentration was 0.56 μM); the volume of probe SEQ ID NO: 21 was 0.5 μL (concentration was 0.4 μM); the volume of probe SEQ ID NO: 24 was 0.8 μL (concentration was 0.64 μM), and the melting curve of the detection results was shown in FIG. Fig.14 As shown, the results showed that dengue virus type I, dengue virus type II, dengue virus type III and dengue virus type IV could be distinguished.

[0120] Comparative Example 2 Detection system composed of probes not modified by LNA

[0121] This comparative example verifies the sample with reference to the reaction system and detection method of Example 2, except that the probes in Table 1 that are not modified with LNA are used. In addition to the above differences, the other materials and process operations used in this comparative example are strictly consistent with those in Example 2. The melting curve of the detection results is shown in FIG. Fig.15 As shown, the results show that the characteristic peaks of the melting curve of the probe without LNA modification appear in the same Tm value range during detection, and dengue virus cannot be typed. In addition, due to the high background fluorescence signal, the characteristic peak starting line is not obvious. In other words, the present invention uses locked nucleic acid modified probes in the melting curve system, which has obvious advantages.

[0122] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition for detecting and typing three mosquito-borne insect-borne viruses, characterized in that: include: The first group for fluorescent probe method: A primer pair and a probe for detecting dengue virus as shown in SEQ ID NOs: 1 to 3; Primer pairs and probes for detecting Zika virus as shown in SEQ ID NOs: 7 to 9; A primer pair and a probe for detecting Chikungunya virus as shown in SEQ ID NOs: 10 to 12; and The second set for melting curve method: A primer pair and a probe for detecting dengue virus type I as shown in SEQ ID NOs: 13 to 15; A primer pair and a probe for detecting dengue virus type II as shown in SEQ ID NOs: 16 to 18; A primer pair and a probe for detecting dengue virus type III as shown in SEQ ID NOs: 19 to 21; The primer pair and probe for detecting dengue virus type IV are shown in SEQ ID NOs: 22-24.

2. The composition according to claim 1, characterized in that The composition also includes an internal standard primer pair and a probe as shown in SEQ ID NOs: 4 to 6.

3. The composition according to claim 1, characterized in that The fluorescent reporter group of the probe for detecting dengue virus as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting Zika virus as shown in SEQ ID NO:9 is ROX; the fluorescent reporter group of the probe for detecting Chikungunya virus as shown in SEQ ID NO:12 is CY5; the fluorescent reporter group of the probe for detecting dengue virus type I as shown in SEQ ID NO:15 is FAM; the fluorescent reporter group of the probe for detecting dengue virus type II as shown in SEQ ID NO:18 is FAM; the fluorescent reporter group of the probe for detecting dengue virus type III as shown in SEQ ID NO:21 is FAM; the fluorescent reporter group of the probe for detecting dengue virus type IV as shown in SEQ ID NO:24 is FAM.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a kit for detecting and typing three mosquito-borne arboviruses, wherein: The three mosquito-borne arboviruses are dengue virus, Zika virus and chikungunya virus.

5. A kit for detecting and typing three mosquito-borne insect-borne viruses, characterized in that: The kit comprises the composition according to any one of claims 1 to 3.

6. The kit according to claim 5, characterized in that In each reaction system, the amount of primer used is 0.05-5 μM, and the amount of probe used is 0.05-5 μM.

7. The kit according to claim 5 or 6, characterized in that The concentration range of the probe for detecting dengue virus type I as shown in SEQ ID NO:15 is 0.16-0.4 μM, the concentration range of the probe for detecting dengue virus type II as shown in SEQ ID NO:18 is 0.24-0.56 μM, the concentration range of the probe for detecting dengue virus type III as shown in SEQ ID NO:21 is 0.08-0.4 μM, and the concentration range of the probe for detecting dengue virus type IV as shown in SEQ ID NO:24 is 0.24-0.64 μM.

8. The kit according to any one of claims 5 to 7, characterized in that The kit also includes a negative quality control product and a positive quality control product.

9. The kit according to any one of claims 5 to 8, characterized in that The kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, a reverse transcriptase, a DNA polymerase, dNTPs (T) and Mg 2+ At least one of .

10. A method for detecting and typing three mosquito-borne arboviruses for non-diagnostic purposes, characterized in that: The method comprises the following steps: (1) extracting or releasing nucleic acid from the sample to be tested; (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition according to any one of claims 1 to 3 or the kit according to any one of claims 5 to 9; (3) Obtain and analyze the results.

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