Kit for detecting West Nile virus, Zika virus and yellow fever virus by one-step method
By designing kits with specific primers and fluorescent probes, a highly efficient and low-cost one-step quantitative PCR method for detecting West Nile virus, Zika virus, and yellow fever virus has been achieved. This solves the problem of the difficulty in simultaneously detecting multiple mosquito-borne viruses in existing technologies, and supports early monitoring and epidemic trend analysis of mosquito-borne viruses.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-30
- Publication Date
- 2026-03-10
AI Technical Summary
Existing detection technologies are insufficient for simultaneously and efficiently detecting West Nile virus, Zika virus, and yellow fever virus at low cost, especially in the early monitoring and epidemiological trend analysis of viruses in mosquito-borne vectors.
A kit containing specific primers and fluorescent probes was designed to simultaneously perform one-step quantitative real-time PCR detection of West Nile virus, Zika virus, and yellow fever virus. Multiple detection is achieved using primer pairs II, III, and IV and fluorescent probes I, II, III, and IV, and the specificity and sensitivity of the detection are ensured through conserved region design.
It achieves high sensitivity and specificity for the detection of West Nile virus, Zika virus and yellow fever virus, and can obtain stable fluorescence signals at a sample concentration of 250 copies/mL, supporting early monitoring and epidemic trend analysis of mosquito-borne viruses.
Smart Images
Figure CN121629084A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of diagnostics for West Nile virus, Zika virus, and yellow fever virus, and more particularly relates to a kit for one-step detection of WNV, ZIKV, and YFV. Background Technology
[0002] West Nile virus (WNV) is a zoonotic virus transmitted by mosquitoes (mainly Culex mosquitoes). It can spread across regions via bird migration, maintaining regional epidemics through a bird-mosquito-bird mechanism. It infects humans or mammals such as horses and sheep through mosquito bites. In humans, 20% develop West Nile fever, characterized by fever, headache, fatigue, body aches, nausea, and vomiting, occasionally accompanied by a rash (on the trunk) and swollen lymph nodes. Severe cases can manifest as West Nile encephalitis or meningitis, or West Nile poliomyelitis. In recent years, it has been prevalent in Africa, Europe, the Americas, Australia, the Middle East, and the Indian subcontinent; reports have also been made in my country.
[0003] Zika virus (ZIKV) is a mosquito-borne virus primarily transmitted by Aedes mosquitoes. Most people infected with Zika virus are asymptomatic; those who do experience symptoms typically present with a rash, fever, conjunctivitis, muscle and joint pain, malaise, and headache, lasting 2-7 days. A large-scale epidemic occurred in the Americas in 2015-2016, and infection during pregnancy can lead to microcephaly and other birth defects, premature birth, and miscarriage. To date, cases of Zika virus infection have been reported in 89 countries and territories.
[0004] Yellow fever virus (YFV) is typically transmitted by vectors such as mosquitoes, ticks, or other arthropods. It usually causes mild symptoms such as fever, headache, and joint pain. Severe cases can lead to jaundice, bleeding, high fever, shock, and organ failure, with a mortality rate of up to 9%. YFV is mainly prevalent in tropical regions such as South America and Africa. Globally, 80,000 to 200,000 people are infected annually, with 30,000 to 78,000 deaths.
[0005] Due to factors such as global warming, accelerated urbanization, and rapid development of tourism and trade, the incidence of mosquito-borne infectious diseases worldwide is on the rise. The prevalence areas of existing diseases are expanding, the frequency of disease outbreaks is changing, and multiple mosquito-borne pathogens may coexist in the same region, posing new challenges to the prevention and control of mosquito-borne viruses.
[0006] Currently, compared to the widely used single / double quantitative PCR detection techniques, multiplex quantitative PCR technology offers greater efficiency and reduces labor costs. Using this kit to monitor the activity of vector-borne viruses such as WNV, ZIKV, and YFV in mosquito populations allows for early detection of viral outbreaks, understanding of epidemic trends, and provides a scientific basis for evaluating control effectiveness and developing control strategies. It also helps predict and control the spread of these diseases. Furthermore, it can provide a basis for clinical diagnosis and treatment of patients.
[0007] Therefore, a kit is needed that can simultaneously detect WNV, ZIKV, and YFV. Summary of the Invention
[0008] To address the above problems, this invention provides a kit for simultaneously detecting West Nile virus, Zika virus, and yellow fever virus, comprising primer pair II for detecting West Nile virus, primer pair III for detecting Zika virus, and primer pair IV for detecting yellow fever virus; wherein,
[0009] The primer pairs II contain primer sequences as shown in SEQ ID NO:4 and 5, respectively;
[0010] The primer pair III comprises upstream primer III-1, upstream primer III-2 and downstream primer III, the sequences of which are shown in SEQ ID NO:7-9, respectively;
[0011] The primer pairs IV contain primer sequences as shown in SEQ ID NO:11 and 12, respectively.
[0012] In one specific embodiment, the kit further includes primer pair I for detecting the mosquito vector internal reference rspL40, wherein the primer pair I contains primer sequences as shown in SEQ ID NO:1 and 2, respectively;
[0013] In one specific embodiment, the kit is a real-time PCR kit and includes a fluorescent probe I for detecting the mosquito vector internal reference rspL40, a fluorescent probe II for detecting West Nile virus, a fluorescent probe III for detecting Zika virus, and a fluorescent probe IV for detecting yellow fever virus.
[0014] The sequence of fluorescent probe I is shown in SEQ ID NO:3, the sequence of fluorescent probe II is shown in SEQ ID NO:6, the sequence of fluorescent probe III is shown in SEQ ID NO:10, and the sequence of fluorescent probe IV is shown in SEQ ID NO:13.
[0015] In one specific embodiment, the fluorescent probes I, II, III and IV are labeled with different fluorescent groups.
[0016] In one specific implementation, all primers in primer pairs I-IV are mixed and dissolved in a primer premix solution, wherein the concentration of each primer in the primer premix solution is 5 μM, and the final working concentration is 0.1 μM.
[0017] In one specific embodiment, the fluorescent probes I-IV are mixed and dissolved in a probe premix solution, wherein the concentration of each probe in the probe premix solution is 5 μM, and the final working concentration is 0.2 μM.
[0018] In one specific implementation, the kit also includes a positive control.
[0019] In one specific implementation, the positive controls include nucleic acid constructs containing West Nile virus genes, nucleic acid constructs containing Zika virus genes, and nucleic acid constructs containing yellow fever virus genes.
[0020] The kit of the present invention can simultaneously perform one-step real-time fluorescence PCR detection of West Nile virus, Zika virus and yellow fever virus. The kit has high sensitivity and specificity, and a stable and effective fluorescence signal can be obtained when the copy number of the fragment in the sample reaches 250 copies / mL. Attached Figure Description
[0021] Figure 1 The amplification curves of rspL40 (mosquito vector internal control) at different annealing extension temperatures with a linearized plasmid template of 5×10^4 copies and final concentrations of upstream and downstream primers and probe of 0.2 μM.
[0022] Figure 2 The amplification curves of WNV at different annealing extension temperatures are shown when the linearized plasmid template has 5×10^4 copies and the final concentrations of upstream and downstream primers and probe are 0.2 μM.
[0023] Figure 3 The amplification curves of ZIKV at different annealing extension temperatures are shown when the linearized plasmid template has 5×10^4 copies and the final concentrations of upstream and downstream primers and probe are 0.2 μM.
[0024] Figure 4 The amplification curves of YFV at different annealing extension temperatures are shown when the linearized plasmid template has 5×10^4 copies and the final concentrations of upstream and downstream primers and probe are 0.2 μM.
[0025] Figure 5 The linearized plasmid template for rspL40 (mosquito vector internal reference) is 5×10. 4Amplification curves at different primer concentrations with copies, a final probe concentration of 0.1 μM, and an annealing extension temperature of 58 °C.
[0026] Figure 6 WNV was performed using a linearized plasmid template of 5 × 10⁻⁶. 4 Amplification curves at different primer concentrations with copies, a final probe concentration of 0.1 μM, and an annealing extension temperature of 58 °C.
[0027] Figure 7 ZIKV was used in a linearized plasmid template of 5 × 10⁻⁶. 4 Amplification curves at different primer concentrations with copies, a final probe concentration of 0.1 μM, and an annealing extension temperature of 58 °C.
[0028] Figure 8 YFV was used in a linearized plasmid template of 5 × 10⁻⁶. 4 Amplification curves at different primer concentrations with copies, a final probe concentration of 0.1 μM, and an annealing extension temperature of 58 °C.
[0029] Figure 9 The linearized plasmid template for rspL40 (mosquito vector internal reference) is 5×10. 1 Amplification curves at different primer concentrations with copies, a final probe concentration of 0.1 μM, and an annealing extension temperature of 58 °C.
[0030] Figure 10 WNV was performed using a linearized plasmid template of 5 × 10⁻⁶. 1 Amplification curves at different primer concentrations with copies, a final probe concentration of 0.1 μM, and an annealing extension temperature of 58 °C.
[0031] Figure 11 ZIKV was used in a linearized plasmid template of 5 × 10⁻⁶. 1 Amplification curves at different primer concentrations with copies, a final probe concentration of 0.1 μM, and an annealing extension temperature of 58 °C.
[0032] Figure 12 YFV was used in a linearized plasmid template of 5 × 10⁻⁶. 1 Amplification curves at different primer concentrations with copies, a final probe concentration of 0.1 μM, and an annealing extension temperature of 58 °C.
[0033] Figure 13 The linearized plasmid template for rspL40 (mosquito vector internal reference) is 5×10. 4 Amplification curves at different probe concentrations when copies, final upstream and downstream primer concentrations are 0.1 μM, and annealing extension temperature is 58 °C.
[0034] Figure 14 WNV was performed using a linearized plasmid template of 5 × 10⁻⁶. 4 Amplification curves at different probe concentrations when copies, final upstream and downstream primer concentrations are 0.1 μM, and annealing extension temperature is 58 °C.
[0035] Figure 15 ZIKV was used in a linearized plasmid template of 5 × 10⁻⁶. 4 Amplification curves at different probe concentrations when copies, final upstream and downstream primer concentrations are 0.1 μM, and annealing extension temperature is 58 °C.
[0036] Figure 16 YFV was used in a linearized plasmid template of 5 × 10⁻⁶. 4 Amplification curves at different probe concentrations when copies, final upstream and downstream primer concentrations are 0.1 μM, and annealing extension temperature is 58 °C.
[0037] Figure 17 The linearized plasmid template for rspL40 (mosquito vector internal reference) is 5×10. 1 Amplification curves at different probe concentrations when copies, final upstream and downstream primer concentrations are 0.1 μM, and annealing extension temperature is 58 °C.
[0038] Figure 18 WNV was performed using a linearized plasmid template of 5 × 10⁻⁶. 1 Amplification curves at different probe concentrations when copies, final upstream and downstream primer concentrations are 0.1 μM, and annealing extension temperature is 58 °C.
[0039] Figure 19 ZIKV was used in a linearized plasmid template of 5 × 10⁻⁶. 1 Amplification curves at different probe concentrations when copies, upstream and downstream primer final concentrations are 0.1 μM respectively, and annealing extension temperature is 58℃.
[0040] Figure 20 YFV was used in a linearized plasmid template of 5 × 10⁻⁶. 1 Amplification curves at different probe concentrations when copies, final upstream and downstream primer concentrations are 0.1 μM, and annealing extension temperature is 58 °C.
[0041] Figure 21 The amplification curves of rspL40 (mosquito vector internal reference) at different linearized plasmid concentrations with final upstream and downstream primer concentrations of 0.1 μM, final probe concentration of 0.2 μM, and annealing extension temperature of 58 °C are shown.
[0042] Figure 22The amplification curves of WNV at different linearized plasmid concentrations are shown when the final concentrations of upstream and downstream primers are 0.1 μM, the final concentration of probe is 0.2 μM, and the annealing extension temperature is 58 °C.
[0043] Figure 23 The amplification curves of ZIKV at different linearized plasmid concentrations are shown when the final concentrations of upstream and downstream primers are 0.1 μM, the final concentration of probe is 0.2 μM, and the annealing extension temperature is 58 °C.
[0044] Figure 24 The amplification curves of YFV at different linearized plasmid concentrations are shown when the final concentrations of upstream and downstream primers are 0.1 μM, the final concentration of probe is 0.2 μM, and the annealing extension temperature is 58 °C.
[0045] Figure 25 This image shows a one-step quantitative PCR assay for rspL40 (mosquito vector internal control), WNV, ZIKV, and YFV. It contains one plasmid template of 5 × 10⁻⁶. 4 The PCR system included three positive controls, three negative controls, and three blank controls. At this point, the final concentrations of the upstream and downstream primers were 0.1 μM, and the final concentration of the probe was 0.2 μM. The annealing extension temperature was 58℃. Detailed Implementation
[0046] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.
[0047] 1. Reagent kit design
[0048] Based on all gene sequences of WNV, ZIKV, and YFV, alignment analysis was performed to identify conserved regions, and primers and probes were designed targeting these conserved sequences. Primer 5 software was used to analyze the designed primers and probes, selecting one or more pairs of primers and probes that would not form hairpin structures or primer dimers. The BLAST function in NCBI was then used to screen for the most specific primer and probe pair, and the sequences were subsequently sent to a biotechnology company for synthesis.
[0049] The designed primer and probe sequences are shown in Table 1.
[0050] Table 1 Primer and Sequence List
[0051]
[0052]
[0053] Positive plasmids were synthesized and prepared from homologous regions of rspL40 (mosquito vector internal reference), WNV, ZIKV, and YFV sequences. After linearization and recovery, the concentration was determined using an ultra-micro spectrophotometer, and the copy number was calculated. The plasmids were then diluted to 4 × 10⁻⁶. 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 Copies / μL, different linearized plasmids of the same concentration and equal volume were mixed as templates for multiplex quantitative PCR.
[0054] For animal tissue homogenate samples or blood samples, extract 200 μL for RNA extraction. Follow the instructions for the RNA extraction kit. Dissolve the RNA precipitate in 50 μL of RNase-free ddH2O.
[0055] In addition, the synthesized primer powder was dissolved in RNase-free ddH2O to a concentration of 100 μM. All upstream and downstream primers were mixed and diluted in equal proportions, with each primer having a final concentration of 10 μM. The synthesized probe powder was dissolved in RNase-free ddH2O to a concentration of 100 μM. All probes were mixed and diluted in equal proportions, with each probe having a final concentration of 10 μM. Further dilutions and additions were made as needed.
[0056] The one-step multiplex real-time 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.
[0057] The one-step multiplex quantitative PCR procedure is as follows: 42℃ for 25 min; 95℃ for 1 min; denaturation at 95℃ for 10 s; 58℃ for 1 min, with simultaneous collection of fluorescence signals, and amplification for 45 cycles.
[0058] Figure 1-4 The corresponding vectors are rspL40 (mosquito vector internal control), WNV, ZIKV, and YFV, with a linearized plasmid template of 5 × 10⁻⁶. 4Amplification curves at different annealing and extension temperatures for copies. The final concentrations of upstream and downstream primers and probe in the PCR system were 0.2 μM. Analysis shows that 58℃ is a relatively ideal annealing and extension temperature, at which ideal Ct values and amplification curves can be obtained.
[0059] Figure 5-12 The figures show amplification curves of rspL40 (mosquito vector internal control), WNV, ZIKV, and YFV at different final primer concentrations at low to medium copy numbers. The final probe concentration in the PCR system was 0.1 μM. Figure 5-8 The linearized plasmid template is 5 × 10 4 copies, Figure 9-12 The linearized plasmid template is 5 × 10 1 Copies. The annealing extension temperature is 58℃. Analysis shows that 0.1 μM is a relatively ideal final concentration of upstream and downstream primers, at which a relatively ideal Ct value and amplification curve can be obtained.
[0060] Figure 13-20 The figures show amplification curves of rspL40 (mosquito vector internal control), WNV, ZIKV, and YFV at different final probe concentrations at low to medium copy numbers. The final concentrations of the upstream and downstream primers in the PCR system were 0.1 μM, respectively. Figure 13-16 The linearized plasmid template is 5 × 10 4 copies, Figure 17-20 The linearized plasmid template is 5 × 10 1 Copies. The annealing extension temperature is 58℃. Analysis shows that 0.2 μM is a relatively ideal final probe concentration, at which a relatively ideal Ct value and amplification curve can be obtained.
[0061] Figure 21-24 The figures show amplification curves for rspL40 (mosquito vector internal control), WNV, ZIKV, and YFV at different linearized plasmid concentrations. The final concentrations of upstream and downstream primers in the PCR system were 0.1 μM, and the final probe concentration was 0.2 μM. The annealing extension temperature was 58℃. Analysis showed that a stable and effective fluorescence signal could be obtained even with a copy number of 5 in the PCR reaction system.
[0062] Figure 25 This image shows a one-step quantitative PCR assay for rspL40 (mosquito vector internal control), WNV, ZIKV, and YFV. It contains one plasmid template of 5 × 10⁻⁶. 4 The kit included three positive controls, three negative controls, and three blank controls. The final concentrations of the upstream and downstream primers in the PCR system were 0.1 μM, and the final probe concentration was 0.2 μM. The annealing extension temperature was 58℃. The results showed that this kit has good detection discrimination and sensitivity.
[0063] 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 principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A kit for simultaneous detection of West Nile virus, Zika virus and Yellow fever virus, characterized in that, comprises primer pair II for detecting West Nile virus, primer pair III for detecting Zika virus, and primer pair IV for detecting yellow fever virus; wherein, the primer pair II comprises primer sequences as shown in SEQ ID NO: 4 and 5, respectively; the primer pair III comprises upstream primer III-1, upstream primer III-2, and downstream primer III, and the sequences are as shown in SEQ ID NO: 7-9, respectively; the primer pair IV comprises primer sequences as shown in SEQ ID NO: 11 and 12, respectively.
2. The kit of claim 1, wherein primer pair I for detecting mosquito-borne internal reference rspL40 is further included, and the primer pair I comprises primer sequences as shown in SEQ ID NO: 1 and 2, respectively.
3. The kit of claim 2, wherein The kit is a fluorescent quantitative PCR kit, and comprises fluorescent probe I for detecting mosquito-borne internal reference rspL40, fluorescent probe II for detecting West Nile virus, fluorescent probe III for detecting Zika virus, and fluorescent probe IV for detecting yellow fever virus. The sequence of the fluorescent probe I is as shown in SEQ ID NO: 3, the sequence of the fluorescent probe II is as shown in SEQ ID NO: 6, the sequence of the fluorescent probe III is as shown in SEQ ID NO: 10, and the sequence of the fluorescent probe IV is as shown in SEQ ID NO:
13.
4. The kit of claim 3, wherein The fluorescent groups labeled on the fluorescent probe I, the fluorescent probe II, the fluorescent probe III, and the fluorescent probe IV are different.
5. The kit of claim 4, wherein All primers in the primer pairs I-IV are mixed and dissolved in a primer premix, and the concentration of each primer in the primer premix is 5 μM, and the working final concentration is 0.1 μM.
6. The kit of claim 5, wherein The fluorescent probes I-IV are mixed and dissolved in a probe premix, and the concentration of each probe in the probe premix is 5 μM, and the working final concentration is 0.2 μM.
7. The kit of any one of claims 1-6, wherein, A positive control is further included.
8. The kit of claim 7, wherein The positive control includes a nucleic acid construct containing a West Nile virus gene, a nucleic acid construct containing a Zika virus gene, and a nucleic acid construct containing a yellow fever virus gene.