Kit for detecting and typing respiratory syncytial virus based on microfluidic method as well as application and method thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a kit for detecting and typing respiratory syncytial viruses based on a microfluidic method and application and a method thereof. The kit provided by the invention comprises a micro-fluidic chip, the micro-fluidic chip is provided with a reaction hole, and the reaction hole is filled with a PCR synergistic freeze-drying mixture; the PCR synergistic freeze-drying mixture is prepared from trimethylglycine, tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA (Bovine Serum Albumin) and nuclease-free water. The components of the PCR synergistic freeze-drying mixture are optimized, and the respiratory syncytial virus is detected and typed in combination with a microfluidic method, so that the detection sensitivity can be obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a kit for detecting and typing respiratory syncytial virus based on a microfluidics method, and an application and method thereof. Background Art

[0002] Respiratory syncytial virus (RSV), as a pathogen of global public health concern, plays a key role in respiratory infections. RSV infection may pose a serious health threat, especially to infants, the elderly and people with weakened immune function.

[0003] RSV belongs to the genus Pneumovirus of the family Paramyxoviridae. Its virus particles are spherical or filamentous, have an envelope, and the genome is a single-stranded negative-strand RNA. During natural infection, the virus is transmitted through air droplets and close contact, infecting respiratory epithelial cells, triggering an immune response, and causing typical symptoms such as fever, cough, and wheezing. In severe cases, it can also cause bronchiolitis and pneumonia, and even be life-threatening.

[0004] RSV is divided into subtype A (RSV-A) and subtype B (RSV-B) according to the antigenicity of its adhesion protein G. Clinically, RSV typing is of great significance. Accurate typing results can guide the formulation of clinical treatment plans. Since RSV subtypes have different sensitivities to drugs, clear typing allows doctors to choose drugs more specifically, thereby improving the treatment effect. From the perspective of disease prevention and control, monitoring the prevalence of RSV subtypes in different regions and seasons will help public health departments plan prevention and control strategies in advance and rationally allocate medical resources. In addition, the severity and prognosis of RSV infection caused by different subtypes are different. RSV-A infection may lead to more serious conditions, such as a relatively high incidence of complications such as respiratory failure, and RSV-B infection is relatively mild; accurate typing can more accurately assess the patient's prognosis and provide more reliable rehabilitation advice. In the field of vaccine research and development, understanding the main epidemic strains can develop more targeted vaccines and improve the protective effect of vaccines.

[0005] However, RSV typing faces many challenges. RSV has a high mutation rate, its antigenicity is constantly changing, and new variants continue to emerge, making traditional typing methods difficult to cope with. At the same time, mixed infections between different types of RSV and between RSV and other pathogens are not uncommon, which further interferes with the accuracy of typing. In addition, existing detection technologies have deficiencies in sensitivity, specificity, and ease of operation, making it difficult to meet the needs of rapid and accurate clinical typing. At present, commonly used RSV typing methods mainly include serological methods, virus culture methods, and genotyping methods. Serological methods, such as immunofluorescence and enzyme-linked immunosorbent assays, detect viral antigens or antibodies in patient serum through specific antibodies, but this method is easily affected by cross-reactions, has limited sensitivity, and is not effective for samples with low viral loads. The virus culture method requires the virus to be inoculated into cell culture, and the type is determined by observing the cytopathic characteristics and combining serological methods. This method is time-consuming, has a low positive rate, and has strict requirements on sample collection and transportation conditions, and is limited in its application in clinical rapid testing. The genotyping method uses reverse transcription polymerase chain reaction (RT-PCR) and sequencing technology to analyze the differences in viral gene sequences for typing. Although it has high accuracy, the operation process is complicated and costly, and it has strict requirements on laboratory conditions and personnel's technical level. In addition, improper primer design may lead to missed detections.

[0006] Against the above technical background, the emergence of microfluidics has brought new hope for RSV typing. Microfluidics has significant advantages. The first is integration and miniaturization. It can integrate multiple experimental steps on a tiny chip, greatly reducing the amount of samples and reagents. It is easy to carry and provides the possibility for on-site detection. Secondly, microfluidics has the characteristics of high throughput and can process multiple samples at the same time, greatly improving the detection efficiency and enabling rapid typing of a large number of samples. Furthermore, it has high sensitivity and can effectively enrich and detect low concentrations of viruses and reduce the missed detection rate. In addition, microfluidics can also shorten the reaction time and achieve rapid detection, which is conducive to rapid clinical diagnosis and treatment. Moreover, this technology has the advantage of being customizable, and can flexibly design chip structure and detection process according to different detection needs.

[0007] Currently, the technology for RSV typing using microfluidics is extremely scarce. Therefore, developing this technology has become a key focus in the current scientific research field. Summary of the invention

[0008] In view of the above shortcomings, the present invention provides a kit for detecting and typing respiratory syncytial virus based on microfluidics, and its application and method. The kit provided by the present invention includes a microfluidic chip, and the microfluidic chip is provided with a reaction well, and the reaction well is filled with a PCR synergistic lyophilized mixture; the PCR synergistic lyophilized mixture is composed of trimethylglycine, tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water. The present invention can significantly improve the detection sensitivity by optimizing the components of the PCR synergistic lyophilized mixture and combining the microfluidics method to detect and type respiratory syncytial virus.

[0009] The technical solution of the present invention is: In the first aspect, the present invention provides a kit for detecting and typing respiratory syncytial virus based on the microfluidics method, the kit comprising a microfluidics chip, the microfluidics chip being provided with reaction wells, the reaction wells being filled with a PCR enhancing lyophilized mixture; the PCR enhancing lyophilized mixture is composed of trimethylglycine, tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water.

[0010] Specifically, the PCR enhancing lyophilized mixture includes 0.5-1.5M trimethylglycine, 0.1-0.3M tetramethylammonium chloride, 2-5U / mL bovine thrombin, 0.1-0.5%v / v NP-40, 10-15%w / v trehalose and 0.02-0.05%w / v BSA.

[0011] Preferably, the PCR enhancing lyophilized mixture comprises 1.0 M trimethylglycine, 0.2 M tetramethylammonium chloride, 5 U / mL bovine thrombin, 0.3% v / v NP-40, 12% w / v trehalose and 0.04% w / v BSA.

[0012] Specifically, the reaction wells are also filled with PCR premix and primer-probe combination.

[0013] Preferably, the PCR premix includes PCR buffer, MgSO 4 , dNTPs and Taq enzyme.

[0014] Specifically, the reaction wells include an RSV-A reaction well, an RSV-B reaction well and an internal reference reaction well.

[0015] Preferably, the primer-probe combination in the RSV-A reaction well is a RSV-A primer-probe combination, which consists of an RSV-A upstream primer, an RSV-A downstream primer and an RSV-A probe.

[0016] In certain embodiments, the RSV-A upstream primer has a nucleotide sequence as shown in SEQ ID NO.1; the RSV-A downstream primer has a nucleotide sequence as shown in SEQ ID NO.2; and the RSV-A probe has a nucleotide sequence as shown in SEQ ID NO.3.

[0017] Preferably, the primer-probe combination in the RSV-B reaction well is a RSV-B primer-probe combination, which consists of an RSV-B upstream primer, an RSV-B downstream primer and an RSV-B probe; In certain embodiments, the RSV-B upstream primer has a nucleotide sequence as shown in SEQ ID NO.4; the RSV-B downstream primer has a nucleotide sequence as shown in SEQ ID NO.5; and the RSV-B probe has a nucleotide sequence as shown in SEQ ID NO.6.

[0018] Preferably, the primer-probe combination in the internal reference reaction well is an internal reference gene primer-probe combination, which consists of a B2M upstream primer, a B2M downstream primer and a B2M probe.

[0019] In certain embodiments, the B2M upstream primer has a nucleotide sequence as shown in SEQ ID NO.7; the B2M downstream primer has a nucleotide sequence as shown in SEQ ID NO.8; and the B2M probe has a nucleotide sequence as shown in SEQ ID NO.9.

[0020] Specifically, the kit also includes a positive control substance or a negative control substance.

[0021] Preferably, the positive control substance is a positive control plasmid containing RSV-A type G gene, RSV-B type G gene and B2M gene.

[0022] Preferably, the negative control substance is nucleic acid-free plum water.

[0023] Specifically, the microfluidic chip in the kit is obtained by filling the PCR enhancement freeze-dried mixture, PCR premix and primer probe combination into reaction wells and then freeze-drying.

[0024] Preferably, the volume ratio of the PCR enhancement freeze-dried mixture, the PCR premix and the primer-probe combination is 6.5:6.5:2.

[0025] In a second aspect, the present invention provides use of the above-mentioned kit in detecting respiratory syncytial virus or typing respiratory syncytial virus.

[0026] Preferably, the respiratory syncytial virus is typed as RSV-A and RSV-B.

[0027] In a third aspect, the present invention provides a method for detecting respiratory syncytial virus or typing respiratory syncytial virus, wherein the method comprises using the above-mentioned kit.

[0028] Specifically, the method comprises the following steps: S1. Take the sample to be tested, extract genomic DNA, and obtain the nucleic acid sample to be tested; S2, injecting the nucleic acid sample to be tested into the injection hole, and flowing it into each reaction hole through the injection channel, so that the nucleic acid sample to be tested reacts with the filler in the reaction hole; S3, performing nucleic acid amplification; S4. Reading and judging the results.

[0029] Preferably, the filler in the reaction well is a freeze-dried product of a PCR enhancement freeze-dried mixture, a PCR premix and a primer-probe combination.

[0030] Preferably, the nucleic acid amplification conditions include: reaction at 50°C for 20 min; pre-denaturation at 95°C for 5 min; reaction at 95°C for 15 s, reaction at 60°C for 30 s, and 45 cycles.

[0031] Preferably, the conditions for determining the result include: The conditions for determining it as RSV-A type: FAM channel Ct value ≤ 35, and the amplification curve shows a typical S-shape; VIC channel Ct value ≤ 35, and the amplification curve shows a typical S-shape; ROX channel Ct value ≥ 35 or no Ct value.

[0032] The conditions for determining it as RSV-B type: FAM channel Ct value ≥ 35 or no Ct value; VIC channel Ct value ≤ 35, and the amplification curve shows a typical S shape; ROX channel Ct value ≤ 35, and the amplification curve shows a typical S shape.

[0033] The conditions for determining a negative result are: FAM channel Ct value ≥ 35 or no Ct value; VIC channel Ct value ≤ 35, and the amplification curve presents a typical S-shape; ROX channel Ct value ≥ 35 or no Ct value.

[0034] Conditions for determining an invalid result: VIC channel Ct value ≥ 35 or no Ct value.

[0035] The beneficial effects of the present invention are as follows: the present invention optimizes the components of the PCR enhanced freeze-dried mixture and combines the microfluidics method to detect and type respiratory syncytial virus, thereby significantly improving the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The test result is positive for RSV-A.

[0037] Figure 2The test result is positive for RSV-B. DETAILED DESCRIPTION

[0038] The present invention will be further explained clearly and completely by examples below. The following examples are only part of the embodiments of the present invention and are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following examples are all routine experiments unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. The microfluidic chip and real-time detector are both products of Beijing Baikangxin Biotechnology Co., Ltd.

[0039] Example 1 A kit for detecting and typing respiratory syncytial virus based on microfluidics 1. Filling and freeze-drying of microfluidic chips 1.1 PCR Enhanced Lyophilized Mixture The PCR synergistic lyophilized mixture is composed of trimethylglycine, tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water. The PCR synergistic lyophilized mixture is nuclease-free water containing 1.0M trimethylglycine, 0.2M tetramethylammonium chloride, 5U / mL bovine thrombin, 0.3%v / v NP-40, 12%w / v trehalose and 0.04%w / v BSA.

[0040] 1.2 Preparation of PCR master mix Tris-HCl buffer, MgSO 4 , dNTPs, Taq enzyme and nuclease-free water were mixed evenly according to the volumes described in Table 1 to prepare the PCR premix.

[0041] Table 1 PCR premix

[0042] 1.3 Primer-probe combinations Primers and probes for RSV-A and RSV-B were designed based on the specific conserved regions of the G gene of RSV-A and RSV-B in the GenBank database; human β-2 microglobulin (B2M) was selected as the internal reference gene, and primers and probes for B2M were designed. The primer and probe sequence information is shown in Table 2.

[0043] Table 2 Primer and probe sequence information

[0044] Note: "F" in the table stands for forward primer, "R" stands for reverse primer, and "P" stands for probe.

[0045] The invention of the present invention is to optimize the PCR synergistic lyophilized mixture, and to determine the respiratory syncytial virus and its typing based on the above primer probe. The above primer probe can be replaced by a primer probe commonly used in the prior art. The above primer probe combination is not used to limit the composition of the primer probe in the microfluidic chip or kit of the present invention.

[0046] According to the above primer and probe sequences, RSV-A primer probe combination, RSV-B primer probe combination and B2M primer probe combination were prepared according to the compositions shown in Tables 3-5.

[0047] Table 3 Composition of RSV-A primer-probe combination

[0048] Table 4 Composition of RSV-B primer-probe combination

[0049] Table 5 Composition of the B2M primer-probe combination

[0050] 1.4 Filling of reaction wells and freeze-drying (1) Add 6.5 μL of PCR enhancer freeze-dried mixture, 6.5 μL of PCR premix and 2 μL of RSV-A primer-probe combination to the RSV-A reaction well; add 6.5 μL of PCR enhancer freeze-dried mixture, 6.5 μL of PCR premix and 2 μL of RSV-B primer-probe combination to the RSV-B reaction well; add 6.5 μL of PCR enhancer freeze-dried mixture, 6.5 μL of PCR premix and 2 μL of B2M primer-probe combination to the internal reference reaction well.

[0051] (2) Place the microfluidic chip in a freeze dryer with a pre-freezing temperature of -40°C to -80°C. After pre-freezing, evacuate the chip and raise the temperature to -20°C to 0°C to allow ice crystals to sublime directly into water vapor. After sublimation drying, further raise the temperature to 20°C-30°C to remove the remaining bound water. After freeze drying, package the chip to obtain the freeze-dried microfluidic chip.

[0052] 2. Positive and negative controls The kit of the present invention also includes a positive control substance and a negative control substance. A positive control plasmid containing RSV-A type G gene, RSV-B type G gene and B2M gene was synthesized by Bioengineering (Shanghai) Co., Ltd. as a positive control substance; and nucleic acid-free plum water was used as a negative control substance.

[0053] Example 2 Method for using a kit for detecting and typing respiratory syncytial virus 1. Sample collection and purification Extraction of nucleic acid samples to be tested: DNA / RNA extraction of samples was performed using the DNA / RNA extraction kit (Yisheng Biotechnology, 19321ES50) instructions. The extracted DNA / RNA can be tested immediately or stored in a -80°C refrigerator for subsequent testing.

[0054] 2. Loading samples into microfluidic chip Take the freeze-dried microfluidic chip prepared in Example 1, inject the nucleic acid sample to be tested into the injection hole, and flow it into each reaction well through the injection channel (10 μL of nucleic acid sample to be tested in each well), and then seal the entire chip with a sealing layer to ensure that there are no bubbles.

[0055] 3. Nucleic Acid Amplification After the sample addition is completed, nucleic acid amplification is performed, and the amplification reaction procedure is shown in Table 6.

[0056] Table 6 Amplification reaction procedure

[0057] 4. Result determination The result determination method is shown in Table 7.

[0058] Table 7 Result determination method

[0059] Example 3 Kit for detecting and typing respiratory syncytial virus based on microfluidics for determination of RSV-A virus and RSV-B virus genomic DNA Select RSV-A virus and RSV-B virus positive samples, extract genomic DNA of the two positive samples. Use the kit described in Example 1 of the present invention and the method described in Example 2 to measure the two nucleic acid samples.

[0060] RSV-A positive test results Figure 1 As shown, the positive test result of RSV-B type is as follows Figure 2 The test results show that the kit provided by the present invention can be successfully used for the determination and differentiation of RSV-A and RSV-B.

[0061] Example 4 Sensitivity of a kit for detecting and typing respiratory syncytial virus based on microfluidics The positive control plasmid containing RSV-A type G gene, RSV-B type G gene and B2M gene was diluted to the equivalent of 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2The samples with 10 copies / mL and 10 copies / mL were measured using the kit described in Example 1 of the present invention and the method described in Example 2. The measurement results are shown in Table 8.

[0062] Table 8 Sensitivity test results

[0063] Note: "+" in the table represents positive, and "-" represents negative.

[0064] The results showed that the minimum detection limit of the kit of the present invention for the two detection targets can reach 10 2 copies / mL, good sensitivity.

[0065] Example 5 A kit for detecting and typing respiratory syncytial virus based on microfluidics for clinical sample testing Patient samples (30 cases) and RSV-B patient samples (26 cases) that have been clinically confirmed as RSV-A type, and healthy volunteer samples (20 cases) were used as test samples, and the kit prepared in Example 1 was used to perform the test according to the method described in Example 2. The test results are shown in Table 9.

[0066] Table 9 Clinical sample test results

[0067] Comparative Example 1 A kit for detecting and typing respiratory syncytial virus based on microfluidics The difference between the kit of comparative example 1 and the kit of embodiment 1 of the present invention is only that: 1.1 The PCR enhancing lyophilized mixture is different.

[0068] The PCR synergistic lyophilized mixture of Comparative Example 1 is composed of trimethylglycine, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water. The PCR synergistic lyophilized mixture is nuclease-free water containing 1.2M trimethylglycine, 5U / mL bovine thrombin, 0.3%v / vNP-40, 12%w / vtrehalose and 0.04%w / v BSA.

[0069] Comparative Example 2 A kit for detecting and typing respiratory syncytial virus based on microfluidics The difference between the kit of comparative example 2 and the kit of embodiment 1 of the present invention is only that: 1.1 The PCR enhancing lyophilized mixture is different.

[0070] The PCR synergistic lyophilized mixture of Comparative Example 2 is composed of tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water. The PCR synergistic lyophilized mixture is nuclease-free water containing 1.2M tetramethylammonium chloride, 5U / mL bovine thrombin, 0.3%v / vNP-40, 12%w / vtrehalose and 0.04%w / v BSA.

[0071] Comparative Example 3 A kit for detecting and typing respiratory syncytial virus based on microfluidics The difference between the kit of comparative example 3 and the kit of embodiment 1 of the present invention is only that: 1.1 The PCR enhancing lyophilized mixture is different.

[0072] The PCR synergistic lyophilized mixture of Comparative Example 3 is composed of trimethylglycine, tetramethylammonium chloride, bovine thrombin, TritonX-100, trehalose, BSA and nuclease-free water. The PCR synergistic lyophilized mixture is nuclease-free water containing 1.0M trimethylglycine, 0.2M tetramethylammonium chloride, 5U / mL bovine thrombin, 0.3%v / v Triton X-100, 12%w / v trehalose and 0.04%w / v BSA.

[0073] Comparative Example 4 A kit for detecting and typing respiratory syncytial virus based on microfluidics The difference between the kit of comparative example 4 and the kit of embodiment 1 of the present invention is only that: 1.1 The PCR enhancing lyophilized mixture is different.

[0074] The PCR synergistic lyophilized mixture of Comparative Example 4 is composed of trimethylglycine, tetramethylammonium chloride, NP-40, trehalose, BSA and nuclease-free water. The PCR synergistic lyophilized mixture is nuclease-free water containing 1.0M trimethylglycine, 0.2M tetramethylammonium chloride, 0.3%v / v NP-40, 12%w / v trehalose and 0.04%w / v BSA.

[0075] Comparative Example 5 A kit for detecting and typing respiratory syncytial virus based on microfluidics The difference between the kit of comparative example 5 and the kit of embodiment 1 of the present invention is only that: 1.1 The PCR enhancing lyophilized mixture is different.

[0076] The PCR synergistic lyophilized mixture of Comparative Example 5 is composed of trimethylglycine, NP-40, trehalose, BSA and nuclease-free water. The PCR synergistic lyophilized mixture is nuclease-free water containing 1.2M trimethylglycine, 0.3% v / v NP-40, 12% w / v trehalose and 0.04% w / v BSA.

[0077] Experimental Example 1 Sensitivity Verification of the Kits of Comparative Examples 1 to 5 The positive control plasmid containing RSV-A type G gene, RSV-B type G gene and B2M gene was diluted to the equivalent of 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 The kits of Comparative Examples 1 to 5 were used to perform the assay according to the method described in Example 2. The assay results are shown in Table 10.

[0078] Table 10 Comparative Example 1-Comparative Example 5 Kit Sensitivity Test Results

[0079] Note: "+" in the table represents positive, and "-" represents negative.

[0080] The results showed that the kits prepared in Comparative Examples 1 to 5 had lower sensitivity than that in Example 1. Among them, Comparative Example 5 had the lowest sensitivity, with the lowest detection limit of RSV-A and RSV-B being only 10 5 copies / mL.

[0081] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A kit for detecting and typing respiratory syncytial virus based on microfluidics, characterized in that: The test kit comprises a microfluidic chip, which is provided with reaction wells, and the reaction wells are filled with a PCR synergistic lyophilized mixture; the PCR synergistic lyophilized mixture consists of trimethylglycine, tetramethylammonium chloride, bovine thrombin, NP-40, trehalose, BSA and nuclease-free water.

2. The kit according to claim 1, characterized in that The PCR enhancement freeze-dried mixture comprises 0.5-1.5M trimethylglycine, 0.1-0.3M tetramethylammonium chloride, 2-5U / mL bovine thrombin, 0.1-0.5%v / v NP-40, 10-15%w / v trehalose and 0.02-0.05%w / v BSA.

3. The kit according to claim 1, characterized in that The PCR enhancement freeze-dried mixture includes 1.0M trimethylglycine, 0.2M tetramethylammonium chloride, 5U / mL bovine thrombin, 0.3%v / v NP-40, 12%w / v trehalose and 0.04%w / v BSA.

4. The kit according to claim 1, characterized in that The reaction wells are also filled with PCR premix and primer-probe combination.

5. The kit according to claim 4, characterized in that The PCR premix solution includes PCR buffer, MgSO4, dNTPs and Taq enzyme.

6. The kit according to claim 4, characterized in that The reaction wells include RSV-A reaction well, RSV-B reaction well and internal reference reaction well; The primer-probe combination in the RSV-A reaction well is a RSV-A primer-probe combination, which consists of an RSV-A upstream primer, an RSV-A downstream primer and an RSV-A probe; The primer-probe combination in the RSV-B reaction well is a RSV-B primer-probe combination, which consists of an RSV-B upstream primer, an RSV-B downstream primer and an RSV-B probe; The primer-probe combination in the internal reference reaction well is an internal reference gene primer-probe combination, which consists of a B2M upstream primer, a B2M downstream primer and a B2M probe.

7. The kit according to claim 1, characterized in that The kit also includes a positive control substance or a negative control substance.

8. Use of the kit according to any one of claims 1 to 7 in detecting respiratory syncytial virus or typing respiratory syncytial virus, characterized in that: The application described is for non-disease diagnosis purposes.

9. The use according to claim 8, characterized in that: The respiratory syncytial virus is classified into RSV-A type and RSV-B type.

10. A method for detecting or typing respiratory syncytial virus, characterized in that: The method comprises using the kit according to any one of claims 1 to 7, and the method is for non-disease diagnosis purposes.

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