Detection reagents and applications for chikungunya virus nucleic acid typing

Abstract

This invention provides a detection reagent for chikungunya virus nucleic acid genotyping and its application. Three sets of specific primers and probes are designed for the three genotypes of chikungunya virus, effectively avoiding cross-interference and improving detection accuracy. The PCR reaction solution, enzyme solution, and primers / probes are premixed to achieve rapid detection in a single tube. Furthermore, by optimizing the PCR reaction solution formulation and adding diatomaceous earth nanoparticles and NP-40, the stability degradation after premixing the reaction solution and enzyme solution is overcome, allowing the fully premixed system to be stably stored, significantly extending the reagent's shelf life and amplification efficiency.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to a rapid detection reagent for the genotyping of Chikungunya virus nucleic acid and its application. Background Technology

[0002] Chikungunya virus (CHIKV) is a single-stranded positive-sense RNA virus belonging to the genus *Alphavirus* of the family Occultviridae. It is the pathogen that causes Chikungunya fever. This virus is found in tropical Africa and Asia and is transmitted to humans through the bites of mosquitoes of the genus *Aedes*, such as *Aedes albopictus* and *Aedes aegypti*. Humans and primates are the main hosts of Chikungunya virus, with acute-phase patients, asymptomatic carriers, and infected primates being the main sources of infection. Chikungunya virus causes symptoms such as fever, rash, and joint pain, similar to dengue fever and Zika virus, making it easily misdiagnosed. Although the mortality rate is low, it can easily lead to large-scale outbreaks and epidemics in areas with high mosquito density.

[0003] Chikungunya virus can be classified into three genotypes based on its sequence: West African, Central-Eastern-Southern African (ECSA), and Asian. These three genotypes differ in gene sequence and geographical distribution. The Asian genotype mainly spreads in Asia, with outbreaks reported in Southeast Asia and other countries and regions. The ECSA is prevalent in tropical and subtropical regions of Africa and Asia. The African genotype is more prevalent on the African continent, with some African countries and regions being its main transmission areas. During transmission, these different subtypes differ in their transmission characteristics and pathogenicity due to variations in ecological environment, host species, and mosquito preferences, directly affecting mosquito transmission routes and the extent of their spread. For example, the Asian genotype is generally less virulent, while the ECSA is more efficient in transmission and more virulent, potentially causing more severe disease symptoms. These differences directly influence the choice of clinical diagnosis and treatment. Currently, imported cases are the main source of infection in my country, but the risk of a large-scale outbreak in the future cannot be ignored. Therefore, there is an urgent need for a method for rapid on-site detection and typing of the chikungunya virus in order to quickly curb its spread and trace its origin and transmission route through typing, thus providing a scientific basis for formulating regional prevention and control measures.

[0004] Common methods for detecting chikungunya virus include real-time quantitative PCR (qPCR), virus isolation, serological testing, antigen detection, and genome sequencing. Virus isolation yields the most accurate results, but takes 1-2 weeks and must be performed in a biosafety level 3 laboratory, placing high demands on time and location. Serological testing requires a large blood sample, takes 2-3 days to obtain results, and other related viral infections may lead to false positives. Antigen detection requires a certain amount of viral particles in the patient sample, making it difficult to detect potential patients. Genome sequencing is time- and costly. qPCR, through the design of primer pairs and probes, amplifies the chikungunya virus-specific gene sequence, requires fewer blood samples, and is less time-consuming, making it suitable for rapid on-site detection of chikungunya virus.

[0005] However, existing qPCR kits and published patents only detect chikungunya virus and cannot identify the three genotypes, which is not conducive to tracing the source of the virus and monitoring its transmission route. Summary of the Invention

[0006] To address the technical problem that existing commercially available chikungunya virus detection reagents cannot type the West African, Central-East-Southern African, and Asian types of the virus, our team has conducted in-depth research and numerous experiments, successfully screening primer-probe combinations that target the three genotypes of chikungunya virus. This allows for simultaneous detection and typing of the chikungunya virus, offering greater clinical significance.

[0007] The first aspect of the present invention provides a detection reagent for chikungunya virus nucleic acid typing, comprising a PCR reaction solution, the PCR reaction solution comprising amplification primer pairs and probes for detecting West African type virus, Central-East-Southern African type virus and Asian type chikungunya virus typing;

[0008] The nucleotide sequences of the amplification primer pairs and probes for the West African type of Chikungunya virus are shown in SEQ ID NO:1-3; the nucleotide sequences of the amplification primer pairs and probes for the Central-East-Southern African type of Chikungunya virus are shown in SEQ ID NO:4-6; and the nucleotide sequences of the amplification primer pairs and probes for the Asian type of Chikungunya virus are shown in SEQ ID NO:7-9.

[0009] The PCR reaction solution also includes diatomaceous earth nanoparticles and NP-40, wherein the particle size of the diatomaceous earth nanoparticles is 50-100 nm.

[0010] In some specific embodiments, the primer-probe combination for chikungunya virus nucleic acid typing also includes an amplification primer pair and a probe for an internal control, wherein the internal control protein is GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The amplification primer pair for the internal control includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO:10, the nucleotide sequence of the reverse primer is shown in SEQ ID NO:11, and the nucleotide sequence of the probe is shown in SEQ ID NO:12.

[0011] In some specific embodiments, the probe is an oligonucleotide fluorescent probe, with a fluorescent reporter group labeled at the 5' end and a fluorescent quencher group labeled at the 3' end. The fluorescent reporter group is selected from FAM, Texas Red / ROX, Cy5, Cy5.5, or HEX / VIC, and the fluorescent reporter groups of any two probes are different; the fluorescent quencher group is selected from one or more of BHQ1, BHQ2, or BHQ3.

[0012] In some specific embodiments, the PCR reaction solution also includes DNA polymerase, dNTPs, and Mg. 2+ and buffer solution.

[0013] Optionally, in the PCR reaction solution: the concentration of the forward primer for West African chikungunya virus nucleic acid amplification is 200-500 nM, the concentration of the reverse primer is 200-500 nM, and the concentration of the probe is 100-200 nM; the concentration of the forward primer for Central-East-South African chikungunya virus nucleic acid amplification is 200-500 nM, the concentration of the reverse primer is 200-500 nM, and the concentration of the probe is 100-200 nM; the concentration of the forward primer for Asian chikungunya virus nucleic acid amplification is 200-500 nM, the concentration of the reverse primer is 200-500 nM, and the concentration of the probe is 100-200 nM.

[0014] Optionally, in the PCR reaction solution, the concentration of the DNA polymerase is 0.2-0.4 U / µL, the concentration of the dNTPs is 1-1.5 mM, and the concentration of Mg... 2+ The concentration of the buffer solution is 3-4 mM, and the concentration of the buffer solution is 10-20 mM.

[0015] Optionally, the concentration of the diatomaceous earth is 1-2 ng / µL, and the concentration of NP-40 is 1-3 ng / µL.

[0016] In some specific embodiments, the buffer solution for the PCR reaction can be at least one of Tris-HCl buffer and HEPES buffer.

[0017] A second aspect of the present invention provides the application of the detection reagents described above in the preparation of Chikungunya virus nucleic acid typing products.

[0018] The beneficial effects of this invention are as follows: (1) Three sets of specific primers and probes are designed for the three genotypes of Chikungunya virus, which do not interfere with each other in the same reaction system and do not produce cross-reactions, thus having excellent detection sensitivity. (2) By premixing the qPCR reaction solution, enzyme solution, and primers and probes, a one-tube rapid detection method is achieved, enabling rapid completion of the amplification reaction and obtaining detection results, effectively improving the ease of operation and the immediacy of detection. (3) The PCR reaction solution formulation is optimized, and diatomaceous earth nanoparticles and NP-40 are innovatively added to the reaction system. The synergistic effect of the two effectively overcomes the problem of decreased stability after premixing the reaction solution and enzyme solution, allowing the fully premixed system to be stably stored, extending the shelf life of the reagents and improving the amplification efficiency. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The results of the sensitivity test for nucleic acid amplification of the Central-East-Southern African Chikungunya virus in Example 3 are shown in the figure. The red curve is the internal control signal curve, and the blue curve is the amplification curve of the Central-East-Southern African Chikungunya virus sample.

[0021] Figure 2 The results of the sensitivity test for nucleic acid amplification of West African Chikungunya virus in Example 3 are shown in the figure. The red curve is the internal reference signal curve and the green curve is the amplification curve of West African Chikungunya virus sample.

[0022] Figure 3 The results of the sensitivity test for the amplification of Asian-type Chikungunya virus nucleic acid in Example 3 are shown. The red curve is the internal control signal curve, and the orange curve is the amplification curve of the Asian-type Chikungunya virus sample.

[0023] Figure 4 The results of the specificity test for Chikungunya virus nucleic acid amplification in Example 3 are shown. The red curve is the internal control signal curve, and the orange, blue and green curves are the amplification curves of three types of Chikungunya virus samples, respectively. The remaining virus samples did not show effective amplification.

[0024] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them.

[0026] It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. All nucleic acid samples used are from clinical sources. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0027] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0028] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0029] The terms "approximately" and "generally" in this invention refer to a range of accuracy that, as would be understood by those skilled in the art, still guarantees the technical effects of the discussed features. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0030] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0031] Furthermore, the terms first, second, third, (1), (2), (3), and the like in the specification and claims are used to distinguish similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and the embodiments described in this invention can be implemented in a different order than that described or illustrated in this invention.

[0032] The following are definitions of some of the terms used in this invention:

[0033] The primer pair described in this invention includes a forward primer (-F) and a reverse primer (-R) for PCR amplification.

[0034] The primer-probe set described in this invention includes amplification primer pairs and probes (-P) for PCR amplification.

[0035] The primer and probe set of this invention includes amplification primer pairs and probes for detecting West African, Central-Eastern-Southern African, and Asian chikungunya virus typing, specifically including:

[0036] (1) First primer and probe set: Amplification primer pair and probe for West African Chikungunya virus, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO:1, the nucleotide sequence of the reverse primer is shown in SEQ ID NO:2, and the nucleotide sequence of the probe is shown in SEQ ID NO:3.

[0037] (2) Second primer and probe set: Amplification primer pairs and probes for the Central-East-South African type of Chikungunya virus, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO:4, the nucleotide sequence of the reverse primer is shown in SEQ ID NO:5, and the nucleotide sequence of the probe is shown in SEQ ID NO:6;

[0038] (3) Third primer and probe set: Amplification primer pairs and probes for Asian type Chikungunya virus, wherein the nucleotide sequence of the forward primer is shown in SEQ ID NO:7, the nucleotide sequence of the reverse primer is shown in SEQ ID NO:8, and the nucleotide sequence of the probe is shown in SEQ ID NO:9.

[0039] In a preferred embodiment, the probe is modified with a fluorescent group and a quenching group. The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quenching group. The fluorescent reporter group is selected from FAM, ROX, Cy5, or VIC, and the fluorescent reporter groups of any two probes are different. The fluorescent quenching group is selected from one or more of BHQ1, BHQ2, or BHQ3.

[0040] In a preferred embodiment, the primer-probe set further includes amplification primer pairs and probes targeting an internal reference protein, which is GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

[0041] In a preferred embodiment, the nucleotide sequences of the primer pairs and probe sets are shown in Table 1 below.

[0042] Table 1

[0043] Primers / Probes Serial Number sequence West African type-F SEQ ID NO.1 CGCGTCCTTTACCAAG West African type-R SEQ ID NO.2 CCAAATTGTCCCG West African type-P SEQ ID NO.3 CAGCCTGGACACCTTT Central-East-Southern Africa type-F SEQ ID NO.4 GAACACGTAACA Central-East-Southern Africa type-R SEQ ID NO.5 GTAATCAAGCGATAG Central-East-Southern Africa type-P SEQ ID NO.6 ACCGTATAAGACTCTA Asian type - F SEQ ID NO.7 AATAGCAACAAAC Asian type - R SEQ ID NO.8 TGCACCGCACAC Asian type - P SEQ ID NO.9 CCCTACGGCGCAGTTCA Internal Reference-F SEQ ID NO.10 GTAGACCCCTTGAA Internal Reference-R SEQ ID NO.11 ACTTTATTGATGG Internal Reference - P SEQ ID NO.12 TCCCTAGGCCCCTC

[0044] The primer-probe set of this invention contains multiple probes and primers with different modifications and fluorescent labels. The purpose of these modifications is to minimize the background signal value of the probes and improve detection sensitivity. In the same reaction system, the primer-probe set of this invention does not interfere with each other and does not produce cross-reactions. Each target in the primer-probe set of this invention has corresponding primers and probes, resulting in higher sensitivity compared to methods using universal primers. Those skilled in the art will readily understand that when the primer-probe set includes amplification primer pairs and probes for distinguishing two or more viral subtypes, the fluorescent reporter groups of any two probes are different.

[0045] The kit of the present invention includes a PCR reaction solution, which includes the primer and probe set described above.

[0046] In a preferred embodiment, the PCR reaction solution further includes DNA polymerase, dNTPs, and Mg. 2+ The ingredients include buffer, diatomaceous earth nanoparticles, and NP-40, with the diatomaceous earth nanoparticles having a particle size of 50-100 nm.

[0047] In some preferred embodiments, the buffer is selected from at least one of Tris-HCl (tris(hydroxymethyl)aminomethane-hydrochloric acid) buffer and HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer.

[0048] In some preferred embodiments, the concentration of the forward primer is 200-500 nM, the concentration of the reverse primer is 200-500 nM, the concentration of the probe is 100-200 nM; the concentration of the DNA polymerase is 0.2-0.4 U / µL; the concentration of the dNTP is 1-1.5 mM; and the concentration of the Mg... 2+ The concentration of the reagent is 3-4 mM; the concentration of the buffer is 10-20 mM; the concentration of the diatomaceous earth is 1-2 ng / µL; and the concentration of NP-40 is 1-3 ng / µL. After the reaction solution is prepared, it should be frozen at -20°C and thawed before use.

[0049] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify detailed conditions are generally performed according to conventional conditions as described in *Molecular Cloning: A Laboratory Manual* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommended conditions (e.g., product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available or can be prepared according to literature methods.

[0050] The testing units in Examples 1-4 of this application include: Zhuhai Municipal Center for Disease Control and Prevention, Guangdong Provincial Center for Disease Control and Prevention, and Shunde District Center for Disease Control and Prevention in Foshan City. The positive samples of Chikungunya virus all came from the aforementioned three disease control centers.

[0051] Example 1

[0052] In this embodiment, three pairs of primers and probes were designed for the E1 region of Chikungunya virus to detect three subtypes of Chikungunya virus: West African, Central-East-Southern African, and Asian. The corresponding primer and probe sequences are shown in the table below, and the internal reference protein is GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

[0053] Table 2 Primer and probe sequence information for Example 1

[0054] Primers / Probes Serial Number sequence Fluorescent reporter group Fluorescence quenching group West African type-F SEQ ID NO.1 CGCGTCCTTTACCAAG - - West African type-R SEQ ID NO.2 CCAAATTGTCCCG - - West African type-P SEQ ID NO.3 CAGCCTGGACACCTTT 5'-VIC 3'-BHQ2 Central-East-Southern Africa type-F SEQ ID NO.4 GAACACGTAACA - - Central-East-Southern Africa type-R SEQ ID NO.5 GTAATCAAGCGATAG - - Central-East-Southern Africa type-P SEQ ID NO.6 ACCGTATAAGACTCTA 5'-FAM 3'-BHQ1 Asian type - F SEQ ID NO.7 AATAGCAACAAAC - - Asian type - R SEQ ID NO.8 TGCACCGCACAC - - Asian type - P SEQ ID NO.9 CCCTACGGCGCAGTTCA 5'-ROX 3'-BHQ2 Internal Reference-F SEQ ID NO.10 GTAGACCCCTTGAA - - Internal Reference-R SEQ ID NO.11 ACTTTATTGATGG - - Internal Reference - P SEQ ID NO.12 TCCCTAGGCCCCTC 5'-CY5 3'-BHQ3

[0055] Example 2

[0056] (1) The test kit components of this embodiment include: PCR reaction solution, the specific component formula of which is shown in the table below.

[0057] Table 3 PCR reaction solution formulation

[0058] Components concentration West African type-F 500 mM West African type-R 500 mM West African type-P 200 mM Central-East-Southern Africa type-F 500 mM Central-East-Southern Africa type-R 500 mM Central-East-Southern Africa type-P 200 mM Asian type - F 500 mM Asian type - R 500 mM Asian type - P 200 mM Internal Reference-F 300 mM Internal Reference-R 300 mM Internal Reference - P 100 mM DNA polymerase 0.2 U / µL dNTP 1.0 U / µ Tris-Hcl 20 mM <![CDATA[Mg 2+ ]]> 4.0 mM Diatomaceous earth nanoparticles (80nm in diameter) 0.1% NP-40 0.2%

[0059] (2) This embodiment also provides a method for preparing chikungunya virus nucleic acid samples and PCR reaction systems.

[0060] Nucleic acid sample preparation: Viral nucleic acid is extracted using nucleic acid extraction reagent (catalog number RT-B-200, batch number 5205013, Zhongyuan Huiji Biotechnology Co., Ltd.), or nucleic acid is released using sample release agent (catalog number SRR-1, batch number 5209001, Zhongyuan Huiji Biotechnology Co., Ltd.).

[0061] Recommended reagent loading ratio: PCR reaction solution : nucleic acid sample = 1:1, for example, 10 μL of PCR reaction solution and 10 μL of nucleic acid sample.

[0062] (3) Perform real-time quantitative PCR amplification.

[0063] The PCR program was as follows: 54℃ for 120s, cycle number 1; 95℃ for 60s, cycle number 1; 95℃ for 5s, 55℃ for 30s, cycle number 42.

[0064] (4) Interpretation of PCR results

[0065] The criteria for interpreting the reaction results are: positive: Ct<40, negative: Ct≥40 or NoCt.

[0066] Example 3

[0067] (1) Sensitivity verification

[0068] Positive samples of Chikungunya virus from patients in Central, Eastern and Southern Africa were serially diluted to obtain concentrations of 1: 204,800 copies / mL, 2: 51,200 copies / mL, 3: 12,800 copies / mL, 4: 3,200 copies / mL, 5: 800 copies / mL, and 6: 200 copies / mL. These samples were tested using the reagents and methods described in Example 2, with each concentration tested three times. The results are as follows: Figure 1 As shown.

[0069] Positive samples from patients with West African chikungunya virus were serially diluted to obtain concentrations of 1: 204,800 copies / mL, 2: 51,200 copies / mL, 3: 12,800 copies / mL, 4: 3,200 copies / mL, 5: 800 copies / mL, and 6: 200 copies / mL. These samples were tested using the reagents and methods described in Example 2, with each concentration tested three times. The results are as follows: Figure 2 As shown.

[0070] Positive samples from patients with Asian-type Chikungunya virus were serially diluted to obtain concentrations of 1: 204,800 copies / mL, 2: 51,200 copies / mL, 3: 12,800 copies / mL, 4: 3,200 copies / mL, 5: 800 copies / mL, and 6: 200 copies / mL. These samples were then tested using the reagents and methods described in Example 2. Each concentration was tested three times. The results are as follows: Figure 3As shown.

[0071] According to the test results, the detection reagent protected in this application has a high detection sensitivity of up to 200 copies / mL.

[0072] (2) Specificity verification

[0073] Samples of various mosquito-borne or similarly symptom-similar viruses, including genotype 3 chikungunya virus (such as Zika virus, dengue virus, yellow fever virus, West Nile virus, new Bunyavirus, hemorrhagic fever with renal syndrome virus, and Japanese encephalitis virus), were selected and tested using the detection reagents and methods of Example 2. The experimental results are shown in the table below:

[0074] Table 4. Results of Chikungunya virus specificity assay (Ct).

[0075] Sample number Virus type FAM VIC ROX CY5 1 Yellow fever virus - - - 29.58 2 Japanese encephalitis virus - - - 29.82 3 Chikungunya virus - West African type - 29.63 - 29.87 4 Chikungunya virus - Central-East-Southern Africa type 29.43 - - 28.9 5 Chikungunya virus - Central-East-Southern Africa type 34.4 - - 28.82 6 Yellow fever virus - - - 29.15 7 Yellow fever virus - - - 29.06 8 Hemorrhagic fever with renal syndrome virus - - - 28.3 9 Chikungunya virus - Central-East-Southern Africa type 32.98 - - 29.25 10 West Nile virus - - - 29.43 11 New Bunyavirus - - - 29.32 12 Yellow fever virus - - - 31.12 13 dengue virus - - - 29.26 14 West Nile virus - - - 29.38 15 Chikungunya virus - Asian type - - 35.27 29.13 16 Chikungunya virus - West African type - 34.8 - 34.37 17 Chikungunya virus - Asian type - - 30.01 28.9 18 Hemorrhagic fever with renal syndrome virus - - - 29.05 19 dengue virus - - - 28.8 20 Zika virus - - - 31.54 21 Zika virus - - - 29.43 22 dengue virus - - - 29.7 23 Chikungunya virus - West African type - 33.35 - 29.37 24 Japanese encephalitis virus - - - 28.68 25 Japanese encephalitis virus - - - 29.8 26 New Bunyavirus - - - 29.92 27 Zika virus - - - 30.29 28 Hemorrhagic fever with renal syndrome virus - - - 29.6 29 Chikungunya virus - Asian type - - 32.8 33.92 30 Zika virus - - - 32.95 31 Hemorrhagic fever with renal syndrome virus - - - 32.55 32 Hemorrhagic fever with renal syndrome virus - - - 32.03 33 dengue virus - - - 28.67 34 New Bunyavirus - - - 35.17 35 West Nile virus - - - 33.93

[0076] The test results show that the detection reagent protected in this application has high specificity for the three genotypes of Chikungunya virus (as shown in the attached document). Figure 4 It also shows no cross-reactivity with Zika virus, dengue virus, yellow fever virus, West Nile virus, new Bunyavirus, hemorrhagic fever with renal syndrome virus, or Japanese encephalitis virus.

[0077] Example 4

[0078] (1) The synergistic effect of diatomaceous earth nanoparticles and NP-40 on the long-term stability of PCR reaction solution

[0079] To verify the effects of diatomaceous earth nanoparticles and NP-40 in the PCR reaction solution on long-term storage stability, an accelerated storage experiment was conducted, and the following four sets of experiments were set up for verification:

[0080] Group A: The detection reagents prepared in Example 2;

[0081] Group B: The only difference between this group and the test reagent in Example 2 is that diatomaceous earth nanoparticles and NP-40 were not added to the PCR reaction solution; the rest of the preparation methods are exactly the same.

[0082] Group C: The only difference between this group and the test reagent in Example 2 is that NP-40 was not added to the PCR reaction solution; the rest of the preparation methods are exactly the same.

[0083] Group D: The only difference from the test reagent in Example 2 is that NP-40 was not added to the PCR reaction solution, but an equal amount of Tween-20 was added instead. The rest of the preparation methods are exactly the same.

[0084] The four sets of reagents were stored at 37°C in the dark for 0, 1, 3, 5, and 7 days, respectively. 10 μL of each reagent was aliquoted and then mixed with 10 μL of extracted Chikungunya virus-Central-East-Southern Africa type nucleic acid as a template. Amplification and detection were performed using the detection method described in Example 2. The experimental results are shown in the table below:

[0085] Table 5. Results of Ct test on the stability of the premixed reaction solution.

[0086] Sample number Storage time at 37℃ / day Experimental group (A) Control group 1 (B) Control group 2 (C) Control group 3 (D) 1 0 27.71 27.75 27.72 27.76 2 1 27.72 28.31 27.96 27.93 3 3 27.78 28.8 28.35 28.21 4 5 27.82 29.19 28.87 28.58 5 7 27.92 29.58 29.31 28.94

[0087] The experimental results showed that adding diatomaceous earth nanoparticles and surfactant NP-40 to the reaction system significantly improved the long-term storage stability of the one-tube premixed reaction system compared to group B (without diatomaceous earth nanoparticles and surfactant) and group C (with only diatomaceous earth nanoparticles). Comparing groups A and D (with both diatomaceous earth nanoparticles and surfactant), the addition of NP-40 was more beneficial to reagent stability than Tween-20.

[0088] (2) Effect of diatomaceous earth nanoparticle size on PCR reaction

[0089] To verify the effect of diatomaceous earth nanoparticle size on PCR reaction, the following four sets of experiments were set up, as shown in the table below:

[0090] Table 6 Experimental group settings

[0091] A B C D diatomaceous earth nanoparticle size 10nm 50nm 100nm 200nm

[0092] The only difference between the PCR reaction solution of group AD and that of Example 2 is the particle size of the diatomaceous earth nanoparticles; the rest of the preparation process is exactly the same.

[0093] Nucleic acid was extracted from positive samples of Chikungunya virus with three genotypes, and the samples were tested using the four sets of reagents described above according to the detection method in Example 2. The experimental results are shown in the table below:

[0094] Table 7. Ct results obtained by using reagents containing diatomaceous earth nanoparticles of different particle sizes.

[0095]

[0096] Experimental results show that adding diatomaceous earth nanoparticles with a particle size of 50-100 nm to the reaction system can significantly reduce the Ct value of the PCR reaction and effectively improve the amplification efficiency against triadic chikungunya virus.

[0097] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A detection reagent for chikungunya virus nucleic acid typing, comprising a PCR reaction solution, characterized in that, The PCR reaction solution includes amplification primer pairs and probes for detecting West African chikungunya virus, Central-East-Southern African chikungunya virus and Asian chikungunya virus typing. The nucleotide sequences of the amplification primer pairs and probes for the West African type of Chikungunya virus are shown in SEQ ID NO:1-3. The nucleotide sequences of the amplification primer pairs and probes for the Central-East-Southern African type of Chikungunya virus are shown in SEQ ID NO:4-6. The nucleotide sequences of the amplification primer pairs and probes for the Asian type of Chikungunya virus are shown in SEQ ID NO:7-9. The PCR reaction solution also includes diatomaceous earth nanoparticles and NP-40, wherein the particle size of the diatomaceous earth nanoparticles is 50-100 nm.

2. The detection reagent according to claim 1, characterized in that, The PCR reaction solution also includes an amplification primer pair and probe for the internal control, the nucleotide sequences of which are shown in SEQ ID NO:10-12.

3. The detection reagent according to claim 1 or 2, characterized in that, The probe is an oligonucleotide fluorescent probe, with a fluorescent reporter group labeled at the 5' end and a fluorescent quencher group labeled at the 3' end. The fluorescent reporter group is selected from FAM, ROX, Cy5 or VIC, and the fluorescent reporter groups of any two probes are different. The fluorescent quencher group is selected from one or more of BHQ1, BHQ2 or BHQ3.

4. The detection reagent as described in claim 1 or 2, characterized in that, The PCR reaction solution also includes DNA polymerase, dNTPs, and Mg. 2+ and buffer solution.

5. The detection reagent as described in claim 4, characterized in that, The buffer solution is selected from at least one of Tris-HCl buffer and HEPES buffer.

6. The detection reagent as described in claim 4, characterized in that, The amplification primer pair includes a forward primer and a reverse primer, the concentration of the forward primer is 200-500 nM, the concentration of the reverse primer is 200-500 nM, and the concentration of the probe is 100-200 nM.

7. The detection reagent as described in claim 6, characterized in that, The concentration of the DNA polymerase is 0.2-0.4 U / µL, the concentration of the dNTPs is 1-1.5 mM, and the concentration of Mg... 2+ The concentration is 3-4 mM.

8. The detection reagent as described in claim 6, characterized in that, The concentration of the buffer solution is 10-20 mM.

9. The detection reagent as described in claim 6, characterized in that, The concentration of the diatomaceous earth nanoparticles is 1-2 ng / µL, and the concentration of NP-40 is 1-3 ng / µL.

10. The use of the detection reagent according to any one of claims 1-9 in the preparation of Chikungunya virus nucleic acid typing products.

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