Kit for detecting CHIKV, DENV, SINV and JEV through one-step method

Abstract

The invention relates to a kit capable of simultaneously detecting chikungunya virus (CHIKV), dengue virus (DENV), Sindbis virus (SINV) and Japanese encephalitis virus (JEV). The kit comprises a primer pair I for detecting CHIKV, a primer pair II for detecting DENV, a primer pair III for detecting SINV and a primer pair IV for detecting JEV. The kit disclosed by the invention can be used for simultaneously carrying out one-step fluorescent quantitative PCR detection on CHIKV, DENV, SINV and JEV, the sensitivity and the specificity of the kit are high, and stable and effective fluorescent signals can be obtained when the copy number of fragments in a detection sample reaches 250 copies / mL.

Description

Technical Field

[0001] The invention belongs to the field of inspection and quarantine, and particularly relates to a kit for detecting CHIKV, DENV, SINV and JEV by a one-step method. Background Art

[0002] Chikungunya virus (CHIKV) is an RNA virus belonging to the genus Alphavirus of the family Togaviridae. It is transmitted through mosquito bites. Symptoms usually include fever and severe joint pain. Joint swelling, muscle pain, headache, nausea, fatigue and rash may also occur. Occasionally, complications of the eyes, heart and nervous system have been reported. The symptoms are similar to those of dengue virus and Zika virus infection and are easily misdiagnosed.

[0003] Dengue virus (DENV) belongs to the Flaviviridae family and is transmitted by Aedes aegypti or Aedes albopictus mosquitoes. It can cause dengue fever, which is characterized by sudden onset, high fever, severe headache, muscle and joint pain, nausea, vomiting, and rash. The risk of secondary infection is higher, with symptoms of severe abdominal pain, persistent vomiting, shortness of breath, vomiting blood or blood in the stool, and even death.

[0004] Sindbis virus (SINV) is a positive-strand RNA virus belonging to the genus Alphavirus of the family Togaviridae. It is transmitted by the bites of mosquitoes such as Culex pipiens and Aedes mosquitoes, and its hosts are vertebrates such as birds, livestock and humans. After infection, it can cause fever, rash, arthritis, and encephalitis in humans, and the diseases caused by them are prone to develop into chronic diseases, causing serious harm to human health.

[0005] Japanese encephalitis virus (JEV) belongs to the Flaviviridae family and is transmitted through the bites of Culex mosquitoes, circulating between storage and amplification hosts such as pigs, cattle and other livestock and wading birds. Humans are generally susceptible to the virus, and the initial symptoms of the disease are mainly acute encephalitis symptoms, such as fever, headache, nausea, vomiting, and drowsiness. In severe cases, there are serious symptoms such as impaired consciousness, convulsions or seizures, and respiratory failure. The mortality rate is about 20% to 30%, and nearly half of the cases may leave neurological sequelae.

[0006] The above four viruses are all major arboviruses, widely distributed around the world, with a huge number of cases, posing a serious threat to public health and animal husbandry production, and are the focus of attention of disease control systems, customs departments, and researchers from higher institutions. Efficient, rapid, and accurate routine monitoring and detection of the above viruses, and achieving "early detection and early control" can effectively reduce social costs and protect public health. Summary of the invention

[0007] To solve the above problems, the present invention provides a kit for simultaneously detecting Chikungunya virus (CHIKV), dengue virus (DENV), Sindbis virus (SINV) and Japanese encephalitis virus (JEV), comprising a primer pair I for detecting CHIKV, a primer pair II for detecting DENV, a primer pair III for detecting SINV, and a primer pair IV for detecting JEV; wherein,

[0008] The primer sequences included in the primer pair 1 are shown in SEQ ID NOs: 1 and 2 respectively;

[0009] The primer sequences included in the primer pair II are shown in SEQ ID NOs: 4 and 5 respectively;

[0010] The primer sequences contained in the primer pair III are shown in SEQ ID NOs: 7 and 8 respectively;

[0011] The primer sequences contained in the primer pair IV are shown as SEQ ID NOs: 10 and 11, respectively.

[0012] In a specific embodiment, the kit is a fluorescent quantitative PCR kit, and includes a fluorescent probe I for detecting CHIKV, a fluorescent probe II for detecting DENV, a fluorescent probe III for detecting SINV, and a fluorescent probe IV for detecting JEV;

[0013] The sequence of the fluorescent probe I is shown in SEQ ID NO:3, the sequence of the fluorescent probe II is shown in SEQ ID NO:6, the sequence of the fluorescent probe III is shown in SEQ ID NO:9, and the sequence of the fluorescent probe IV is shown in SEQ ID NO:12.

[0014] In a specific embodiment, the fluorescent probes I, II, III and IV are labeled with different fluorescent groups.

[0015] In a specific embodiment, all primers in the primer pairs I-IV are mixed and dissolved in a primer premix solution, the concentration of each primer in the primer premix solution is 10 μM, and the final working concentration is 0.2 μM.

[0016] In a specific embodiment, the fluorescent probes I-IV are mixed and dissolved in a probe premix solution, the concentration of each probe in the probe premix solution is 15 μM, and the final working concentration is 0.6 μM.

[0017] In a specific embodiment, the kit further comprises a positive control.

[0018] In a specific embodiment, the positive control includes a nucleic acid construct containing a CHIKV gene, a nucleic acid construct containing a DENV gene, a nucleic acid construct containing a SINV gene, and a nucleic acid construct containing a JEV gene.

[0019] The kit of the present invention can simultaneously perform one-step fluorescent quantitative PCR detection on CHIKV, DENV, SINV and JEV. The kit has high sensitivity and specificity, and a stable and effective fluorescent signal can be obtained when the copy number of the fragment in the detection sample reaches 250 copise / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For CHIKV, the plasmid template is 5 × 10 4 Amplification curves at different annealing and extension temperatures when the final concentrations of copies, upstream and downstream primers, and probes were 0.2 μM respectively.

[0021] Figure 2 For DENV, the plasmid template is 5 × 10 4 Amplification curves at different annealing and extension temperatures when the final concentrations of copies, upstream and downstream primers, and probes were 0.2 μM respectively.

[0022] Figure 3 For SINV, the plasmid template is 5 × 10 4 Amplification curves at different annealing and extension temperatures when the final concentrations of copies, upstream and downstream primers, and probes were 0.2 μM respectively.

[0023] Figure 4 For JEV, the plasmid template is 5 × 10 4 Amplification curves at different annealing and extension temperatures when the final concentrations of copies, upstream and downstream primers, and probes were 0.2 μM respectively.

[0024] Figure 5 For CHIKV, the plasmid template is 5 × 10 4 Amplification curves at different primer concentrations when the final probe concentration is 0.1 μM and the annealing extension temperature is 58°C.

[0025] Figure 6 For DENV, the plasmid template is 5 × 10 4 Amplification curves at different primer concentrations when the final probe concentration is 0.1 μM and the annealing extension temperature is 58°C.

[0026] Figure 7 For SINV, the plasmid template is 5 × 10 4Amplification curves at different primer concentrations when the final probe concentration is 0.1 μM and the annealing extension temperature is 58°C.

[0027] Figure 8 For JEV, the plasmid template is 5 × 10 4 Amplification curves at different primer concentrations when the final probe concentration is 0.1 μM and the annealing extension temperature is 58°C.

[0028] Fig. 9 For CHIKV, the plasmid template is 5 × 10 1 Amplification curves at different primer concentrations when the final probe concentration is 0.1 μM and the annealing extension temperature is 58°C.

[0029] Fig.10 For DENV, the plasmid template is 5 × 10 1 Amplification curves at different primer concentrations when the final probe concentration is 0.1 μM and the annealing extension temperature is 58°C.

[0030] Fig.11 For SINV, the plasmid template is 5 × 10 1 Amplification curves at different primer concentrations when the final probe concentration is 0.1 μM and the annealing extension temperature is 58°C.

[0031] Fig.12 For JEV, the plasmid template is 5 × 10 1 Amplification curves at different primer concentrations when the final probe concentration is 0.1 μM and the annealing extension temperature is 58°C.

[0032] Fig.13 For CHIKV, the plasmid template is 5 × 10 4 Amplification curves at different probe concentrations when the final concentration of upstream and downstream primers is 0.2 μM and the annealing extension temperature is 58°C.

[0033] Fig.14 For DENV, the plasmid template is 5 × 10 4 Amplification curves at different probe concentrations when the final concentration of upstream and downstream primers is 0.2 μM and the annealing extension temperature is 58°C.

[0034] Fig.15 For SINV, the plasmid template is 5 × 10 4 Amplification curves at different probe concentrations when the final concentration of upstream and downstream primers is 0.2 μM and the annealing extension temperature is 58°C.

[0035] Fig.16For JEV, the plasmid template is 5 × 10 4 Amplification curves at different probe concentrations when the final concentration of upstream and downstream primers is 0.2 μM and the annealing extension temperature is 58°C.

[0036] Fig.17 For CHIKV, the plasmid template is 5 × 10 1 Amplification curves at different probe concentrations when the final concentration of upstream and downstream primers is 0.2 μM and the annealing extension temperature is 58°C.

[0037] Fig.18 For DENV, the plasmid template is 5 × 10 1 Amplification curves at different probe concentrations when the final concentration of upstream and downstream primers is 0.2 μM and the annealing extension temperature is 58°C.

[0038] Fig.19 For SINV, the plasmid template is 5 × 10 1 Amplification curves at different probe concentrations when the final concentration of upstream and downstream primers is 0.2 μM and the annealing extension temperature is 58°C.

[0039] Fig. 20 For JEV, the plasmid template is 5 × 10 1 Amplification curves at different probe concentrations when the final concentration of upstream and downstream primers is 0.2 μM and the annealing extension temperature is 58°C.

[0040] Fig.21 This is the amplification curve of CHIKV at different linearized plasmid concentrations when the final concentration of the upstream and downstream primers is 0.2 μM, the final concentration of the probe is 0.6 μM, and the annealing extension temperature is 58°C.

[0041] Fig. 22 This is the amplification curve of DENV at different linearized plasmid concentrations when the final concentration of the upstream and downstream primers is 0.2 μM, the final concentration of the probe is 0.6 μM, and the annealing extension temperature is 58°C.

[0042] Fig.23 This is the amplification curve of SINV at different linearized plasmid concentrations when the final concentration of the upstream and downstream primers is 0.2 μM, the final concentration of the probe is 0.6 μM, and the annealing extension temperature is 58°C.

[0043] Fig.24 This is the amplification curve of JEV at different linearized plasmid concentrations when the final concentration of upstream and downstream primers is 0.2 μM, the final concentration of the probe is 0.6 μM, and the annealing extension temperature is 58°C.

[0044] Fig.25This is a one-step fluorescence quantitative detection diagram for CHIKV, DENV, SINV, and JEV. It contains a plasmid template of 5×10 0 copies of the positive control, three negative controls, and three blank controls. DETAILED DESCRIPTION

[0045] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0046] 1. Kit Design

[0047] According to all gene sequences of CHIKV, DENV, Sindbis, and JEV, we compared and analyzed and searched for conserved regions, and used Primer 5 software to design primers and probes for conserved sequences, and selected one or several pairs of primers and probes that would not form hairpin structures or primer dimers. We then used the BLAST function in NCBI to screen for the pair of primers and probes with the highest specificity, and then sent the sequence to a biological company for synthesis.

[0048] The designed primer and probe sequences are shown in Table 1.

[0049] Table 1 Primers and sequences

[0050]

[0051]

[0052] The conserved regions of CHIKV, DENV, SINV, and JEV sequences were selected to synthesize and prepare positive plasmids. After linearization and recovery, the concentration was measured using an ultra-micro spectrophotometer, and the copy number was calculated and diluted to 4 × 10 6 copies / μL, 4×10 5 copies / μL, 4×10 4 copies / μL, 4×10 3 copies / μL, 4×10 2 copies / μL, 4×10 1 copies / μL, 4×10 0 Four linearized plasmid templates were mixed in equal volumes as templates for multiplex fluorescence quantitative PCR.

[0053] In addition, add RNase Free dH2O to the synthesized primer powder, and the concentration after dissolution is 100μM. All upstream and downstream primers are mixed and diluted in equal proportions, and the final concentration of each primer is 20μM. Add RNase Free dH2O to the synthesized probe powder, and the concentration after dissolution is 100μM. All probes are mixed and diluted in equal proportions, and the final concentration of each probe is 20μM. Dilute and add according to specific needs later.

[0054] The one-step multiplex fluorescence quantitative PCR system is as follows: 2×One Step RT-PCR Buffer 12.5μL; TaqDNA Polymerases 0.5μL; RT Enzyme 0.5μL; Forward Primer 0.5μL; Reverse Primer 0.5μL; Probe 1μL; Total RNA&DNA 5μL; RNase Free dH2O 4.5μL.

[0055] The one-step multiplex fluorescence quantitative PCR program was as follows: 42°C for 25 min; 95°C for 1 min; denaturation at 95°C for 10 s; 58°C for 1 min, simultaneous collection of fluorescence signals, and amplification for 45 cycles.

[0056] Figure 1-4 They were CHIKV, DENV, SINV, and JEV in the presence of 5×10 4 The amplification curves of different annealing extension temperatures when the number of copies was 100. The final concentrations of upstream and downstream primers and probes in the PCR system were 0.2 μM respectively. The analysis showed that 58°C was the ideal annealing extension temperature, at which the ideal Ct value and amplification curve could be obtained.

[0057] Figure 5-12 The amplification curves of CHIKV, DENV, SINV, and JEV at different primer concentrations at low and medium copy numbers are shown. The final concentration of the probe in the PCR system is 0.1 μM, where Figure 5-8 Plasmid template is 5×10 4 copies, Figure 9-12 Plasmid template is 5×10 1 copies. The annealing extension temperature is 58°C. Analysis shows that 0.2μM is a relatively ideal concentration of upstream and downstream primers, and at this primer concentration, a relatively ideal Ct value and amplification curve can be obtained.

[0058] Figure 13-20 The following are the amplification curves of CHIKV, DENV, SINV, and JEV at different probe concentrations at low and medium copy numbers. The final concentrations of the upstream and downstream primers in the PCR system were 0.2 μM, respectively. Figure 13-16 Plasmid template is 5×104 copies, Figure 17-20 Plasmid template is 5×10 1 The annealing extension temperature is 58°C. Analysis shows that 0.6μM is an ideal probe concentration, at which an ideal Ct value and amplification curve can be obtained.

[0059] Figure 21-24 The amplification curves of CHIKV, DENV, SINV, and JEV at different linearized plasmid concentrations are shown. The final concentrations of upstream and downstream primers in the PCR system were 0.2 μM, and the final concentration of the probe was 0.6 μM. The annealing extension temperature was 58°C. Three replicates were performed at each level. The analysis showed that a stable and effective fluorescence signal could be obtained when the copy number was 5 in the PCR reaction system.

[0060] Fig.25 This is a one-step fluorescence quantitative detection diagram for CHIKV, DENV, SINV, and JEV. It contains a plasmid template of 5×10 0 The test results showed that this kit has good detection discrimination and sensitivity.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for simultaneously detecting Chikungunya virus (CHIKV), dengue virus (DENV), Sindbis virus (SINV) and Japanese encephalitis virus (JEV), characterized in that: It includes a primer pair I for detecting CHIKV, a primer pair II for detecting DENV, a primer pair III for detecting SINV, and a primer pair IV for detecting JEV; wherein, The primer sequences included in the primer pair 1 are shown in SEQ ID NOs: 1 and 2 respectively; The primer sequences included in the primer pair II are shown in SEQ ID NOs: 4 and 5 respectively; The primer sequences contained in the primer pair III are shown in SEQ ID NOs: 7 and 8 respectively; The primer sequences contained in the primer pair IV are shown as SEQ ID NOs: 10 and 11, respectively.

2. The kit according to claim 1, characterized in that The kit is a fluorescent quantitative PCR kit, and includes a fluorescent probe I for detecting CHIKV, a fluorescent probe II for detecting DENV, a fluorescent probe III for detecting SINV, and a fluorescent probe IV for detecting JEV; The sequence of the fluorescent probe I is shown in SEQ ID NO:3, the sequence of the fluorescent probe II is shown in SEQ ID NO:6, the sequence of the fluorescent probe III is shown in SEQ ID NO:9, and the sequence of the fluorescent probe IV is shown in SEQ ID NO:

12.

3. The kit according to claim 2, characterized in that The fluorescent probes I, II, III and IV are labeled with different fluorescent groups.

4. The kit according to claim 3, characterized in that All primers in the primer pairs I-IV are mixed and dissolved in a primer premix solution, the concentration of each primer in the primer premix solution is 10 μM, and the final working concentration is 0.2 μM.

5. The kit according to claim 4, characterized in that The fluorescent probes I-IV are mixed and dissolved in a probe premix solution, the concentration of each probe in the probe premix solution is 15 μM, and the final working concentration is 0.6 μM.

6. The kit according to any one of claims 1 to 5, characterized in that A positive control was also included.

7. The kit according to claim 6, characterized in that The positive control includes a nucleic acid construct containing CHIKV gene, a nucleic acid construct containing DENV gene, a nucleic acid construct containing SINV gene and a nucleic acid construct containing JEV gene.

Images