Primer probe set for combined detection of multiple rodent-derived viruses and digital droplet PCR (polymerase chain reaction) detection kit

Abstract

The invention relates to the technical field of biological detection, and provides a primer probe set for combined detection of multiple rodent-derived viruses and a digital droplet PCR detection kit. The primer probe set comprises a hantaan virus detection PCR primer probe, a first virus detection PCR primer probe, a new bunyavirus detection PCR primer probe and an internal standard GAPDH gene primer probe, and the sequences are shown as SEQ ID NO.1-12. The digital droplet PCR detection kit comprises the primer probe set provided by the invention. According to the primer probe set and the digital droplet PCR detection kit disclosed by the invention, multiple nucleic acid targets can be effectively and jointly detected and accurately quantified; the kit has the advantages of high throughput, capability of realizing accurate quantification of low-abundance nucleic acid, high detection sensitivity, good repeatability, strong specificity, simplicity and convenience in operation and capability of providing an accurate detection result within 2-3 hours.

Description

Technical Field

[0001] The present invention belongs to the field of medical biological detection technology, relates to the rapid detection of Hantaan virus, Seoul virus and new Bunyavirus, and provides a primer probe set and a digital droplet PCR detection kit for the joint detection of multiple rodent-derived viruses. Background Art

[0002] Rodents are the natural hosts of many highly pathogenic viruses and are the natural hosts of many pathogens such as Hantaan virus (HTNV), Seoul virus (SEOV) and new Bunyavirus (SFTSV). These viruses are mainly transmitted through contact or aerosols after their excrement contaminates the environment, and can cause human diseases such as hemorrhagic fever with renal syndrome, Hantavirus pulmonary syndrome and fever with thrombocytopenia syndrome.

[0003] Traditional detection methods (such as virus isolation, immunological testing, and qPCR) are limited in sensitivity, convenience, and multiplex detection capabilities. Digital PCR, a next-generation nucleic acid quantification technology, achieves absolute single-molecule quantification through partitioned amplification. Compared to qPCR, it offers higher sensitivity and specificity and eliminates the need for standard curves, making it more suitable for rapid, simple, and multi-pathogen detection. Currently, digital droplet PCR has not been used to detect the aforementioned viruses. Summary of the Invention

[0004] In order to address the shortcomings of traditional detection methods in sensitivity, throughput and convenience, the present invention has designed and developed a multiple combined detection method based on droplet digital PCR (ddPCR) technology, which can simultaneously achieve high-throughput, high-sensitivity and high-specificity detection of HTNV, SEOV and SFTSV. The technical problem to be solved by the present invention is to break through the limitations of existing rodent-derived virus detection methods and provide a detection system that can complete the absolute quantification of three pathogen nucleic acids in a single reaction. The detection method of the present invention has the advantages of low detection limit (up to 1 copies / μL), strong anti-interference ability, good repeatability, and no need for a standard curve. It can complete sample detection within 3 hours, providing reliable technical support for early diagnosis, epidemic monitoring and precise prevention and control of rodent-derived virus infections.

[0005] In order to achieve the above objectives, the first aspect of the present invention provides a primer probe set for the joint detection of multiple rodent viruses, wherein the primer probe set includes a PCR primer probe for Hantaan virus detection, a PCR primer probe for Seoul virus detection, a PCR primer probe for New Bunyavirus detection, and a primer probe for the internal standard GAPDH gene.

[0006] Furthermore, the primer probe set includes (as shown in Table 1):

[0007] A) Primer pairs and Taqman probes for detecting the Hantaan virus S gene, the nucleotide sequences of which are shown in SEQ ID NO. 1 to SEQ ID NO. 3, respectively;

[0008] B) primer pairs and Taqman probes for detecting the Seoul virus S gene, the nucleotide sequences of which are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively;

[0009] C) primer pairs and Taqman probes for detecting the new bunyavirus L gene, the nucleotide sequences of which are shown in SEQ ID NO. 7 to SEQ ID NO. 9, respectively;

[0010] D) The primer pair and Taqman probe for detecting the internal standard GAPDH gene, the nucleotide sequences of which are shown in SEQ ID NO. 10 to SEQ ID NO. 12, respectively.

[0011] Furthermore, the 5' ends of the four probes contain a fluorescence generating group (such as FAM, Atto425, VIC, ROX, etc.) and the 3' ends contain a fluorescence quenching group (such as BHQ1 and BHQ2, etc.). The fluorescence generating groups connected to the four probes are different.

[0012] Furthermore, the probe sequence (SEQ ID NO. 3) for detecting Hantaan virus was labeled with a FAM fluorescent group at the 5' end and a BHQ1 quenching group at the 3' end;

[0013] The probe sequence for detecting Seoul virus (SEQ ID NO.6) is labeled with Atto425 fluorescent group at the 5' end and BHQ1 quenching group at the 3' end;

[0014] The probe sequence for detecting the new bunyavirus (SEQ ID NO. 9) is labeled with a VIC fluorescent group at the 5' end and a BHQ1 quenching group at the 3' end;

[0015] The 5' end of the probe sequence (SEQ ID NO. 12) for detecting the internal standard GAPDH gene is labeled with a ROX fluorescent group, and the 3' end is labeled with a BHQ2 quenching group.

[0016] The second aspect of the present invention provides use of the primer-probe set described above in preparing a combined detection product for rodent-derived viruses.

[0017] The primer-probe set of the present invention can efficiently identify and accurately quantify Hantaan virus, Seoul virus, and New Bunyavirus at one time, with high detection sensitivity, good repeatability, and strong specificity.

[0018] The Tm values ​​of all probes of the present invention are close to and higher than the Tm values ​​of primers, and the Tm values ​​of primers are also close to the same 53-55° C., ensuring the binding of primers and probes to target sequences during the amplification process.

[0019] As a preferred detection product, the third aspect of the present invention provides a digital droplet PCR detection kit for the joint detection of multiple rodent-derived viruses, which includes multiple digital droplet PCR reactants, and the multiple digital droplet PCR reactants include the primer probe set described above.

[0020] Furthermore, the multiplex digital droplet PCR reactants further include PCR buffer, Taq DNA polymerase, ROX dye, nuclease-free water and / or an exogenous internal standard primer probe pair.

[0021] Furthermore, based on the total volume of the multiplex digital droplet PCR reaction, the concentration of each primer in the multiplex digital droplet PCR reaction ranges from 200 to 1000 nmol / L, and the concentration of the probe ranges from 200 to 400 nmol / L.

[0022] Furthermore, the final concentration of each primer in the multiplex digital droplet PCR reaction is 900 nmol / L, and the final concentration of the probe is 250 nmol / L.

[0023] Furthermore, the kit also includes one or more of an RNA extraction reagent, an RNA reverse transcription reagent, a positive quality control product, and a negative quality control product.

[0024] Furthermore, the positive quality control product is a plasmid containing the specific nucleic acid sequence of each target virus and the exogenous internal standard GAPDH specific nucleic acid fragment; the negative quality control product is nuclease-free water.

[0025] The method for detecting a sample with the kit of the present invention comprises the following steps:

[0026] (1) Sample extraction: The present invention uses plasmids containing specific nucleic acid sequences of each target virus and specific nucleic acid fragments of the exogenous internal standard GAPDH, which are stored in a -80°C refrigerator or tested immediately;

[0027] (2) Preparation of PCR reaction system: The total volume of the PCR reaction system is 15 μL, including 3 μL of PCR buffer, 0.15 μL of Taq DNA polymerase, 0.3 μL of ROX dye, 3.87 μL of digital droplet primer-probe mixture, 1 μL of nucleic acid sample, and nuclease-free water to 15 μL;

[0028] (3) Droplet generation: 14 μL of the PCR reaction solution was transferred to a microfluidic chip and placed in a droplet generator to prepare droplets, thereby generating droplets within the chip.

[0029] (4) PCR amplification: The microfluidic chip with generated droplets was placed in a digital PCR amplifier for PCR amplification. The PCR reaction program was set as follows: 95°C for 5 min; {95°C for 15 s, 56°C for 30 s, 72°C for 1 min}*40 cycles; 28°C for 10 min;

[0030] (5) Chip reading and result analysis and judgment: The microfluidic chip after PCR amplification is placed in a biochip reader. The instrument takes a picture of the microfluidic chip and uses software to analyze the droplet situation and calculate the concentration to obtain the positive copy number concentration of multiple channels. The positive and negative nature of the nucleic acid sample of the sample being tested is determined based on the positive copy number concentration of each channel. The reference standard is:

[0031] Hantaan virus S gene: If FAM ≥ 0.2 copies / μL, the channel is judged as positive; if FAM ≥ 0.1 copies / μL and FAM < 0.2 copies / μL, the channel is reported as suspected positive and requires retesting; if FAM < 0.1 copies / μL in repeated tests, the channel is negative or below the detection limit of this kit; if FAM ≥ 0.1 copies / μL, the channel is reported as positive;

[0032] Seoul virus S gene: If Atto425 ≥ 0.2 copies / μL, the channel is judged as positive; if Atto425 ≥ 0.1 copies / μL and Atto425 < 0.2 copies / μL, the channel is reported as suspected positive and requires retesting; if Atto425 < 0.1 copies / μL in repeated tests, the channel is negative or below the detection limit of this kit; if Atto425 ≥ 0.1 copies / μL, the channel is reported as positive;

[0033] New Bunyavirus L gene: If VIC ≥ 0.2 copies / μL, the channel is judged to be positive; if VIC ≥ 0.1 copies / μL and VIC < 0.2 copies / μL, the channel is reported as suspected positive and requires retesting; if VIC < 0.1 copies / μL in repeated tests, the channel is negative or below the detection limit of this kit; if VIC ≥ 0.1 copies / μL, the channel is reported as positive.

[0034] Digital droplet PCR is a technology for absolute quantification of nucleic acid molecules. It distributes conventional PCR into a large number of tiny reaction droplets, each of which may or may not contain one or more copies of the target nucleic acid molecule. The amplification results are calculated by counting the number of positive reaction units and using statistical methods to calculate the copy number of the target gene in the original sample. Digital droplet PCR has the advantage of direct and independent nucleic acid quantification without relying on control samples and standard curves. In addition, digital droplet PCR does not require detection of the intersection of the fluorescence signal and the threshold, is not dependent on the Ct value, and greatly reduces the impact of PCR reaction inhibitors during the system distribution process.

[0035] The primer probe set and digital droplet PCR detection kit for the combined detection of multiple rodent-derived viruses of the present invention have the following beneficial effects:

[0036] 1. The technical solution of the present invention can effectively and jointly detect, identify and accurately quantify three rodent-derived viruses. After seven-channel digital droplet PCR testing, the present invention has the advantages of high detection throughput, accurate quantification of low-abundance nucleic acids, high detection sensitivity, good repeatability, strong specificity, simple operation, and can provide accurate detection results within 2-3 hours, thereby achieving accurate diagnosis and subsequent precise treatment of patients.

[0037] 2. Exogenous internal reference can ensure the quality control of nucleic acid plasmids during the detection process of nucleic acid extraction and PCR amplification to avoid false negative problems.

[0038] 3. Simultaneous detection of multiple targets has the characteristics of small sample usage, short detection time, high detection throughput, and accurate quantification of low abundance. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 1D graph showing the specificity test results of Hantaan virus (A), Seoul virus (B), and New Bunyavirus (C) under single-plex detection in Example 2;

[0040] Figure 2 2D graphs of the test results for Hantaan virus / New Bunyavirus (A), Hantaan virus / Seoul virus (B), and Seoul virus / New Bunyavirus (C) under multiplex detection in Example 3;

[0041] Figure 3 This is a one-dimensional graph of the test results of the linear relationship between Hantaan virus (A), Seoul virus (B), and New Bunyavirus (C) under multiple detection in Example 3;

[0042] Figure 4 This is a diagram showing the analysis of the linearity test results under multiple detection in Example 3;

[0043] Figure 5This is a one-dimensional graph of the quantitative detection results of actual samples (lung, spleen, liver) of wild animals in Example 4. DETAILED DESCRIPTION

[0044] In the following examples, experimental procedures, where specific conditions are not specified, generally followed conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0045] Example 1: Primer and probe design and screening

[0046] To multiplex detection of different rodent-derived viruses in samples, specific primers and probes must be designed. First, several conserved sequences of viral targets were searched for on the NCBI website and downloaded as FASTA files. The downloaded gene sequences were aligned using Vector NTI software to obtain relatively conserved sequences. This allowed the design of suitable primer and probe locations, as well as the need to avoid highly mutated sites and design degenerate bases. Using the primer design software Primer Express, multiple pairs of primer and probe preparatory sequences were designed based on the sequences of the Hantaan virus S gene, Seoul virus S gene, and New Bunyavirus L gene. First, the specificity of the primers and probes was preliminarily verified online using NCBI-nBLAST. Second, the viruses being tested were RNA viruses, which are unstable and susceptible to RNase A degradation. Loop structures are unstable and easily broken, while stem structures are more stable. This experiment employed the minimum free energy method and predicted the secondary structure of the target genes using RNAfold (http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi). Finally, DNAStar software was used to analyze the complementarity between multiple pairs of primers and probes and the amplified fragments involved. All primers and probes were synthesized by Shanghai Bioengineering.

[0047] Finally, the sequence information of the optimal multiple primers and probes (SEQ ID No. 1 to SEQ ID No. 12) selected through experimental verification is shown in Table 1.

[0048] Table 1 Primer and probe combinations of the present invention

[0049]

[0050]

[0051] F: Forward Primer; R: Reverse Primer; P: Probe.

[0052] Example 2: ddPCR reaction system

[0053] This example provides a specific method for using the kit. The primers and probes shown in the sequence in Table 1 are evenly mixed with a digital droplet PCR kit purchased from Pilot Genetics to obtain a multiplex digital droplet PCR reaction. The concentration of each primer in the multiplex digital droplet PCR reaction is 900 nmol / L, and the concentration of the probe is 250 nmol / L.

[0054] Prepare a PCR reaction solution according to the test requirements. The total volume of the PCR reaction system is 15 μL, including 3 μL of PCR buffer, 0.15 μL of Taq DNA polymerase, 0.3 μL of ROX dye, 3.87 μL of digital droplet primer-probe mix, 1 μL of nucleic acid sample, and nuclease-free water to 15 μL. Transfer 14 μL of the PCR reaction solution to a microfluidic chip and place it in a droplet generator to generate droplets within the chip. The microfluidic chip with droplet generation was placed in a digital PCR amplifier for PCR amplification. The PCR reaction program was set as follows: 95°C for 5 min; {95°C for 15 s, 56°C for 30 s, 72°C for 1 min} x 40 cycles; 28°C for 10 min; and storage at 28°C. Chip reading and result analysis and judgment: The microfluidic chip after PCR amplification is placed in the biochip reader. The instrument takes a picture of the microfluidic chip and uses software to analyze the droplet situation and calculate the concentration to obtain the positive copy number concentration of each channel. The positive or negative nature of the nucleic acid sample of the tested sample is determined based on the positive copy number concentration of each channel.

[0055] In order to determine the specific amplification of single to multiplex ddPCR, the present invention first investigated the amplification of the primer probe of a single substrate, and set up a negative control group and a positive group for detection. Figure 1 As shown, each substrate and the corresponding primer probe can produce positive results and the negative control group without substrate cannot produce positive results. There is a clear difference between the positive droplets and the negative droplets.

[0056] Example 3: Performance analysis of the kit

[0057] (1) Multiple detection performance:

[0058] This study aimed to evaluate the detection capability of a multiplex ddPCR system by simultaneously detecting three different viruses: HTNV, SEOV, and SFTSV in a single reaction. This study used optimized specific primer pairs and probe combinations designed for these three viruses. The experimental results showed that the multiplex ddPCR system was not only able to effectively detect the three viruses simultaneously. Figure 2 As shown, the multiplex ddPCR system can simultaneously detect three viruses in a single reaction system.

[0059] (2) Linear relationship:

[0060] Five viral detection target nucleic acids were diluted to concentrations of 2000, 200, 20, and 2 copies / reaction, respectively, to evaluate the dynamic range. Three replicates were performed for each concentration. 2 Values ​​above 0.98 are considered within the current range. Substrate concentrations are based on singleplex ddPCR quantification, and dilutions are performed to achieve the desired concentration. The X-axis represents the theoretical concentration, and the Y-axis represents the measured concentration. Unit: copies / reaction.

[0061] Depend on Figures 3-4 As shown in the figure, the substrates are linear in the range of 2-2000 copies / reaction, and the theoretical concentration and the detected concentration are linearly regressed, where R 2 Both are 0.9999.

[0062] (3) Sensitivity analysis:

[0063] The five virus detection target nucleic acids were diluted to concentrations of 50, 25, and 10 copies / reaction, respectively. The primer probe set in Example 1 and the digital droplet PCR detection method in Example 2 were used for multiplex ddPCR amplification and detection analysis. 20 replicates were performed, and a positive detection rate of 95% was used as the minimum detection limit (LOD), which is the sensitivity. Based on the LOD evaluation, the limit of quantification (LOQ) is defined as the lowest concentration at which the coefficient of variation (CV) of all replicates is less than 25%. As can be seen from Table 2, the ddPCR method of the present invention has good detection sensitivity.

[0064] Table 2 LOQ and LOD of multiplex ddPCR

[0065]

[0066] a: mean of the test results, b: theoretical concentration, unit: copies / reaction.

[0067] Example 4: Actual sample detection

[0068] Multiplex ddPCR detection was performed on samples collected from Rattus norvegicus according to the primer probe set in Example 1 and the digital droplet PCR detection method in Example 2. Figure 5 Table 3 and Table 4 show the results of nucleic acid extraction from the lung, liver, and spleen tissues of six Rattus norvegicus samples. The test results show that the multiplex ddPCR primer probe set and digital droplet PCR detection kit of the present invention can be well used to detect several common rodent-borne viruses.

[0069] Table 3 Qualitative and quantitative detection results of multiplex ddPCR of wildlife samples

[0070]

[0071] Caption: -: negative.

[0072] The undescribed parts of the present invention are the same as the prior art or are implemented using the prior art. The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned specific implementation methods, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A primer-probe set for the joint detection of multiple rodent-derived viruses, characterized in that: Including PCR primer probes for Hantan virus detection, PCR primer probes for Seoul virus detection, and PCR primer probes for new Bunyavirus detection. The upstream and downstream primer sequences for detecting Hantaan virus are shown in SEQ ID NO. 1 and 2, respectively, and the probe sequence is shown in SEQ ID NO. 3; The upstream and downstream primer sequences for detecting Seoul virus are shown in SEQ ID NOs. 4 and 5, respectively, and the probe sequence is shown in SEQ ID NO. 6; The upstream and downstream primer sequences for detecting the new bunyavirus are shown in SEQ ID NO. 7 and 8, respectively, and the probe sequence is shown in SEQ ID NO.

9.

2. The primer-probe set for joint detection of multiple rodent-derived viruses according to claim 1, characterized in that: Also included are internal reference universal primers and probes. The sequences of the internal reference universal upstream and downstream primers are shown in SEQ ID NOs. 10 and 11, respectively, and the sequence of the internal reference universal probe is shown in SEQ ID NO.

12.

3. The primer-probe set for joint detection of multiple rodent-derived viruses according to claim 2, characterized in that: All probes contain a fluorescence generating group at the 5' end and a fluorescence quenching group at the 3' end. The four probes are connected to different fluorescence generating groups.

4. The primer-probe set for joint detection of multiple rodent-derived viruses according to claim 3, characterized in that: The 5' end of the Hantaan virus detection probe is labeled with a FAM fluorescent group and the 3' end is labeled with a BHQ1 quenching group; The Seoul virus detection probe is labeled with an Atto425 fluorescent group at the 5' end and a BHQ1 quencher at the 3' end; The 5' end of the probe for detecting new bunyaviruses is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ3 quenching group; The 5' end of the universal probe for detecting the internal reference GAPDH is labeled with a ROX fluorescent group, and the 3' end is labeled with a BHQ2 quenching group.

5. Use of the primer-probe set according to any one of claims 1 to 4 in preparing a product for the combined detection of multiple rodent-derived viruses.

6. A digital droplet PCR detection kit for the joint detection of multiple rodent-derived viruses, characterized in that: The method comprises a multiplex digital droplet PCR reaction, wherein the multiplex digital droplet PCR reaction comprises the primer probe set according to any one of claims 1 to 4.

7. The digital droplet PCR detection kit for joint detection of multiple rodent-derived viruses according to claim 6, characterized in that: The concentration range of each primer was 200–1000 nmol / L, and the concentration range of the probe was 200–400 nmol / L; The multiplex digital droplet PCR reactants further include PCR buffer, Taq DNA polymerase, ROX reference dye, nuclease-free water and / or an exogenous internal standard primer probe pair; The kit also includes one or more of an RNA extraction reagent, an RNA reverse transcription reagent, a positive quality control product, and a negative quality control product.

8. The digital droplet PCR detection kit for joint detection of multiple rodent-derived viruses according to claim 7, characterized in that: The final concentration of each primer was 900 nmol / L, and the final concentration of the probe was 250 nmol / L. The positive quality control product is a plasmid containing the specific nucleic acid sequence of each target virus and the specific nucleic acid fragment of the exogenous internal standard GAPDH; the negative quality control product is nuclease-free water.

9. The digital droplet PCR detection kit for joint detection of multiple rodent-derived viruses according to claim 7, characterized in that: The PCR reaction system was prepared as follows: the total volume of the system was 15 μL, including 3 μL of PCR buffer, 0.15 μL of Taq DNA polymerase, 0.3 μL of ROX dye, 3.87 μL of digital droplet primer-probe mixture, 1 μL of nucleic acid sample, and nuclease-free water to 15 μL. The method for generating droplets is as follows: 14 μL of the reaction solution of the PCR reaction system is transferred to a microfluidic chip, which is placed in a droplet generator for droplet preparation to generate droplets in the chip; The PCR reaction program was set as follows: 95°C for 5 min; 40 cycles of {95°C for 15 s, 56°C for 30 s, 72°C for 1 min}; and 28°C for 10 min.

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