Method for detecting virus

Abstract

To provide a new method for detecting a virus by which many kinds of viruses derived from various species can efficiently and accurately be detected in one reaction to improve diagnostic efficiency and provide a reliable detection result.SOLUTION: The present invention provides a virus detection method comprising the steps of: immobilizing virus nucleic acid primer sequences corresponding to viruses derived from various species on a solid-phase support; supplying a specimen sample to the solid-phase support; setting a plurality of amplification reaction conditions; and performing an amplification reaction on the specimen sample supplied to the solid-phase support based on the amplification reaction conditions to simultaneously detect a plurality of viruses derived from various species under the amplification reaction.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for detecting viruses, and in particular to a method for detecting viruses performed on cultured cells, which can be applied in the field of cell therapy. [Background technology]

[0002] Due to the unique nature of cell therapy products, which are primarily composed of living cells, terminal sterilization procedures are not possible, and large-scale purification, virus removal, or virus inactivation steps are generally not possible during the manufacturing process of cell therapy products. Therefore, strict control must be exercised over the sources of the raw materials used to manufacture the therapeutic products, especially the source of the primary cells, and animal-derived materials or reagents. Manufacturers must provide detailed descriptions of the sources of the various raw materials used in the manufacturing process and outline the tests performed on those materials to ensure the safety and quality of the products.

[0003] Controlling the origin and quality of cells is a key step in the manufacturing process of cell therapy products. Cell origin requires extensive management, including collection methods, donor screening, and cell banking systems. Furthermore, allogeneic cells must be screened and tested for specific pathogens, such as human immunodeficiency virus (HIV) and hepatitis virus, depending on the donor. Furthermore, when using animal-derived reagent components, such as bovine or porcine serum or trypsin, the cells must be subjected to relevant viral detection.

[0004] To meet the growing demand for cell therapy, fast and accurate virus detection methods are becoming increasingly important. Conventional virus detection methods can only detect a single virus or a limited number of viruses from the same species, limiting their application in situations involving multiple pathogens and various species. Furthermore, multiplex PCR technology has been widely applied to simultaneously detect many different pathogens. This technology allows for the simultaneous amplification of many target nucleic acids in a single reaction by designing multiple specific amplification primers. However, conventional multiplex PCR technology faces many challenges, including nonspecific binding between amplification primers, differences in amplification efficiency, and difficulty in optimizing reaction conditions. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is an urgent need to develop a novel virus detection method that can efficiently and accurately detect many types of viruses derived from various species in a single reaction, thereby improving diagnostic efficiency and providing reliable detection results. [Means for solving the problem]

[0006] In view of the above, the present invention provides a method for detecting viruses, thereby solving the well-known problems mentioned above.

[0007] The present invention provides a virus detection method that is applicable to detecting multiple viruses at once, and includes the steps of immobilizing viral nucleic acid primer sequences corresponding to each of the viruses derived from various species on a solid phase carrier; supplying a sample to the solid phase carrier; setting multiple amplification reaction conditions including an amplification reaction temperature, an amplification primer length, an amplification primer concentration, and the number of amplification reaction cycles; and performing an amplification reaction on the sample supplied to the solid phase carrier based on the multiple amplification reaction conditions, and detecting multiple viruses derived from various species at once through the amplification reaction.

[0008] In the above detection method, the amplification reaction temperature range is 70°C or lower, the amplification primer length range is 14mer to 30mer, the amplification primer concentration range is 0.1 μM to 1 μM, and the number of amplification reaction cycles is 40 or less.

[0009] In the above-mentioned detection method, the step of setting the plurality of amplification reaction conditions further includes a step of confirming the amplification reaction condition with the highest sensitivity among the plurality of amplification reaction conditions, a step of optimizing the other plurality of amplification reaction conditions by fixing the amplification reaction condition with the highest sensitivity, and a step of setting the amplification reaction condition with the highest sensitivity and the other plurality of amplification reaction conditions after optimization as fixed optimal conditions, and in the step of performing the amplification reaction on the specimen sample supplied to the solid phase carrier based on the plurality of amplification reaction conditions, the amplification reaction is performed by using the fixed optimal conditions.

[0010] In the above-mentioned detection method, the step of setting the plurality of amplification reaction conditions further includes a step of optimizing the amplification reaction condition with the highest sensitivity while fixing the other plurality of amplification reaction conditions after optimization, a step of subsequently optimizing the other plurality of amplification reaction conditions while fixing the amplification reaction condition with the highest sensitivity after optimization, and a step of setting the amplification reaction condition with the highest sensitivity after optimization and the other plurality of amplification reaction conditions after subsequent optimization as the fixed optimal conditions.

[0011] In the above-mentioned detection method, the virus detection method of the present invention further includes the analysis of nucleic acid purification reaction, reverse transcription reaction, polymerase chain reaction and amplification reaction.

[0012] In the above detection method, the plurality of viruses include human RNA viruses, human DNA viruses, bovine DNA viruses, and porcine DNA viruses.

[0013] In the above-mentioned detection method, the human RNA viruses include human immunodeficiency virus type 1 (HIV-I), human immunodeficiency virus type 2 (HIV-II), hepatitis A virus (HAV), hepatitis C virus (HCV), human T-lymphotropic virus type 1 (HTLV-I), and human T-lymphotropic virus type 2 (HTLV-II).

[0014] In the above-mentioned detection method, the human DNA viruses include Hepatitis B Virus (HBV), B19 Parvovirus (Parvovirus B19), Epstein-Barr Virus Type 4 (EBV), Human Cytomegalovirus (HCMV), Human Adenovirus (HAdV), Human Herpesvirus 6 (HHV-6), Human Herpesvirus 7 (HHV-7), Human Herpesvirus 8 (HHV-8), Human Papillomavirus Type 16 (HPV-16), Human Papillomavirus Type 18 (HPV-18), BK virus (BK These include the JC virus and the JC virus.

[0015] In the above detection method, the bovine DNA virus includes bovine polyomavirus (BPyV).

[0016] In the above detection method, the porcine DNA viruses include Porcine Circovirus Type 1 (PCV-1), Porcine Circovirus Type 2 (PCV-2), and Porcine Parvovirus (PPV). [Effects of the Invention]

[0017] In summary, the present invention provides a virus detection method. While conventional techniques only detect viruses of the same species, the present invention immobilizes corresponding viral nucleic acid primer sequences on a solid support and prepares and sets amplification reaction conditions, thereby detecting multiple viruses of different species at once. Furthermore, while conventional techniques use costly and inefficient trial and error methods to prepare and set amplification reaction conditions, the present invention identifies and fixes the amplification reaction conditions with the highest sensitivity and optimizes other amplification reaction conditions, replacing the conventional trial and error method, thereby reducing cost waste and improving virus detection efficiency. Furthermore, while conventional techniques have insufficient virus detection accuracy, the present invention further optimizes the amplification reaction conditions with the highest sensitivity and subsequently optimizes other amplification reaction conditions, thereby improving virus detection accuracy and reducing the probability of false negatives or false positives in virus tests. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a flow chart illustrating the steps of a virus detection method according to a specific embodiment of the present invention. [Figure 2] FIG. 2 is a flow chart illustrating the steps of a virus detection method according to another specific embodiment of the present invention. [Figure 3] FIG. 3 is a flow chart illustrating the steps of a virus detection method according to another specific embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] In order to make the advantages, spirit, and features of the present invention more easily and clearly understood, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that these specific embodiments are merely representative examples of the present invention, and the specific methods, devices, conditions, materials, etc. exemplified therein are in no way used to limit the present invention or the corresponding specific embodiments. In addition, the elements in the drawings are only used to indicate their relative positions and are not drawn based on their actual proportions. The step symbols of the present invention are only used to distinguish different steps, and in no way represent the order of the steps.

[0020] Referring to Figure 1, Figure 1 is a flow chart illustrating the steps of a virus detection method according to a specific embodiment of the present invention. As shown in Figure 1, the virus detection method according to this specific embodiment is applicable to the simultaneous detection of multiple viruses. The virus detection method according to this specific embodiment includes the steps of: immobilizing viral nucleic acid primer sequences corresponding to viruses of various species on a solid support (S1); supplying a sample to the solid support (S2); setting multiple amplification reaction conditions, including amplification reaction temperature, amplification primer length, amplification primer concentration, and amplification reaction cycle number (S3); and amplifying the sample supplied to the solid support according to the amplification reaction conditions (S4), thereby simultaneously detecting multiple viruses of various species through the amplification reaction. The virus detection method according to this specific embodiment further includes nucleic acid purification, reverse transcription, polymerase chain reaction, and amplification reaction analysis.

[0021] In addition, the viruses referred to in the virus detection method in this specific embodiment may include human RNA viruses, human DNA viruses, bovine DNA viruses, and porcine DNA viruses, among which human RNA viruses include human immunodeficiency virus type 1 (HIV-I), human immunodeficiency virus type 2 (HIV-II), hepatitis A virus (HAV), hepatitis C virus (HCV), human T-lymphotropic virus type 1 (HTLV-I), and human T-lymphotropic virus type 2 (HTLV-II). Human DNA viruses include Hepatitis B Virus (HBV), Parvovirus B19, Epstein-Barr Virus Type 4 (EBV), Human Cytomegalovirus (HCMV), Human Adenovirus (HAdV), Human Herpesvirus 6 (HHV-6), Human Herpesvirus 7 (HHV-7), Human Herpesvirus 8 (HHV-8), Human Papillomavirus Type 16 (HPV-16), Human Papillomavirus Type 18 (HPV-18), BK virus, and JC virus. Bovine DNA viruses can include Bovine Polyomavirus (BPyV).Porcine DNA viruses can include porcine circovirus type 1 (PCV-1), porcine circovirus type 2 (PCV-2), and porcine parvovirus (PPV). In practical applications, the types of viruses detected are not limited to these, and the types of viruses can be increased or decreased based on the needs of the manufacturer or experimental design. Furthermore, the source of the virus is not limited to the above-mentioned species, and can also be a plant, animal, or other biological species.

[0022] The solid support in step S1 can be a biological chip such as a protein chip or a gene chip. The specimen sample in step S2 can be a blood sample (e.g., plasma, serum, whole blood, etc.), an airway sample (e.g., saliva, nasal secretions, etc.), or a tissue sample (e.g., tissue section, tissue puncture, etc.). In practice, the specimen sample can be selected based on the needs of the manufacturer. In this specific example, the amplification reaction temperature in step S3 can be 70°C or less, the amplification primer length can be 14-mer to 30-mer, the amplification primer concentration can be 0.1 μM to 1 μM, and the number of amplification reaction cycles can be 40 or less. However, in actual applications, these are not limited to these, and the amplification reaction conditions can be selected based on the manufacturer's needs and experimental design in addition to the parameters mentioned above. Furthermore, the parameter ranges and optimized parameter values ​​of the amplification reaction conditions can be adjusted and designed based on the equipment used by the manufacturer or the experimental requirements. In this specific example, viral nucleic acid primer sequences corresponding to various viruses are immobilized on a solid support, and amplification reaction conditions are prepared and set, thereby achieving the detection of multiple viruses from various species at once.

[0023] In practice, the parameter ranges and optimized parameter values ​​for the amplification reaction conditions corresponding to each virus can be found through multiple replicate trials (try and error). For example, to detect multiple human viruses in a single run, amplification reaction conditions (including amplification reaction temperature, amplification primer length, amplification primer concentration, and number of amplification cycles) must be designed for each virus. In this case, each virus must include at least the four parameter ranges listed above, and optimized parameter values ​​can be obtained by arranging and combining each parameter range. If detection is to be performed for various viruses, more parameter ranges must be added and combined, which may require an increased number of replicate trials. However, replicate trials require a significant amount of time and resources, and effective solutions may not be found quickly, especially when the problem is complex or the number of trials is large. Furthermore, multiple replicate trials may consume a large amount of materials, manpower, and energy, resulting in cost waste, reduced efficiency, and a lack of systematization. Therefore, the following specific examples further illustrate how to efficiently find parameter ranges and optimized parameter values ​​for amplification reaction conditions.

[0024] Referring to FIG. 2, FIG. 2 is a flowchart illustrating the steps of a virus detection method according to another specific embodiment of the present invention. As shown in FIG. 2, this specific embodiment differs from the above-described specific embodiments in that the virus detection method of this specific embodiment includes a step of setting amplification reaction conditions, which may further include steps S31, S32, S33, and S41. The above steps can be performed sequentially following step S2. In step S31, the amplification reaction conditions with the highest sensitivity are identified from among multiple amplification reaction conditions. In step S32, the amplification reaction conditions with the highest sensitivity are fixed, and other amplification reaction conditions are optimized. In step S33, the amplification reaction conditions with the highest sensitivity and the optimized other amplification reaction conditions are set as fixed optimal conditions. In step S41, an amplification reaction is performed on a sample supplied to a solid support based on the amplification reaction conditions. Multiple viruses from various species are simultaneously detected through the amplification reaction using the fixed optimal conditions. In this specific embodiment, multiple amplification reaction conditions must first be set and adjusted before the amplification reaction is performed. If adjusting the parameters of one of the amplification reaction conditions significantly changes the outcome of the amplification reaction, then this amplification reaction condition is the amplification reaction condition with the highest sensitivity.

[0025] For example, during the amplification reaction, various amplification reaction results can be obtained by adjusting parameters such as amplification reaction temperature, amplification primer length, amplification primer concentration, and number of amplification reaction cycles. If it is found during this process that adjusting the amplification reaction temperature can significantly change the amplification reaction results, the amplification reaction temperature parameter is fixed, and other amplification reaction conditions (including amplification primer length, amplification primer concentration, and number of amplification reaction cycles) are further adjusted to find the corresponding optimized parameter values. Subsequently, multiple viruses from various species are detected simultaneously in the amplification reaction using the fixed amplification reaction temperature parameter value and the optimized parameter values ​​of other amplification reaction conditions. This specific example replaces the well-known method of repeated testing by identifying and fixing the amplification reaction conditions with the highest sensitivity and optimizing other amplification reaction conditions, thereby reducing cost waste and improving virus detection efficiency. Note that the other steps in the virus detection method of this specific example are generally the same as the corresponding steps in the previous specific examples, and will not be described in detail again here.

[0026] Furthermore, the step of setting amplification reaction conditions in the virus detection method of the above-mentioned specific embodiment may also include other aspects. Referring to FIG. 3, FIG. 3 is a flowchart illustrating the steps of a virus detection method of another specific embodiment of the present invention. As shown in FIG. 3, the step of setting amplification reaction conditions in the virus detection method of this specific embodiment may further include steps S34, S35, and S36. These steps are performed following step S33. In step S34, the amplification reaction conditions with the highest sensitivity are optimized by fixing other amplification reaction conditions after optimization. In step S35, the amplification reaction conditions with the highest sensitivity after optimization are fixed, and other amplification reaction conditions are subsequently optimized. In step S36, the amplification reaction conditions with the highest sensitivity after optimization and the other amplification reaction conditions after subsequent optimization are set as fixed optimal conditions. Specifically, once optimized parameter values ​​for other amplification reaction conditions (including amplification primer length, amplification primer concentration, and number of amplification reaction cycles) have been found, the parameter value for the amplification reaction temperature, which was initially fixed, can be further optimized in a similar manner. The optimized parameter value for the amplification reaction temperature is then fixed, and other amplification reaction conditions are subsequently optimized. Furthermore, multiple viruses from various species are detected simultaneously in the amplification reaction using the optimized parameter value for the fixed amplification reaction temperature and the subsequently optimized parameter values ​​for the other amplification reaction conditions. In this specific embodiment, further optimizing the amplification reaction conditions with the highest sensitivity and subsequently optimizing the other amplification reaction conditions further improves the accuracy of virus detection and reduces the probability of false negatives or false positives in the test. Note that the other steps in the virus detection method of this specific embodiment are generally the same as the corresponding steps in the previous specific embodiments, and will not be described in detail again here.

[0027] In summary, the present invention provides a virus detection method. While conventional techniques only detect viruses of the same species, the present invention immobilizes corresponding viral nucleic acid primer sequences on a solid support and prepares and sets amplification reaction conditions, thereby detecting multiple viruses of different species at once. Furthermore, while conventional techniques use costly and inefficient trial and error methods to prepare and set amplification reaction conditions, the present invention identifies and fixes the amplification reaction conditions with the highest sensitivity and optimizes other amplification reaction conditions, replacing the conventional trial and error method, thereby reducing cost waste and improving virus detection efficiency. Furthermore, while conventional techniques have insufficient virus detection accuracy, the present invention further optimizes the amplification reaction conditions with the highest sensitivity and subsequently optimizes other amplification reaction conditions, thereby improving virus detection accuracy and reducing the probability of false negatives or false positives in virus tests.

[0028] It is hoped that the detailed description of the preferred embodiment above will more clearly illustrate the features and spirit of the present invention, but the above-mentioned preferred embodiment in no way limits the scope of the present invention. On the contrary, the purpose is to cover various modifications and equivalent adjustments within the scope of the patent application to be filed by the present invention. Therefore, the scope of the patent application to be filed by the present invention should be interpreted in the broadest sense based on the above description, so that this scope will cover all possible modifications and equivalent adjustments. [Explanation of symbols]

[0029] S1 Step S2 Step S3 Step S4 Step S31 Step S32 Step S33 Step S34 Step S35 Step S36 Step S41 Step

Claims

1. It is applied to detecting multiple viruses at once, Immobilizing viral nucleic acid primer sequences corresponding to viruses from various species on a solid support; applying a specimen sample to the solid phase support; Setting a plurality of amplification reaction conditions including an amplification reaction temperature, an amplification primer length, an amplification primer concentration, and an amplification reaction cycle number; and performing an amplification reaction on the specimen sample supplied to the solid phase carrier based on the plurality of amplification reaction conditions, and simultaneously detecting a plurality of viruses derived from various species under the amplification reaction; A virus detection method comprising:

2. 2. The virus detection method of claim 1, wherein the amplification reaction temperature range is 70°C or lower, the amplification primer length range is 14 mer to 30 mer, the amplification primer concentration range is 0.1 μM to 1 μM, and the number of amplification reaction cycles is 40 or less.

3. The step of setting a plurality of amplification reaction conditions includes: identifying the amplification reaction condition with the highest sensitivity among the plurality of amplification reaction conditions; optimizing the other amplification reaction conditions in the plurality of amplification reaction conditions in which the sensitivity is highest; and setting the amplification reaction conditions with the highest sensitivity and the other multiple amplification reaction conditions after optimization as fixed optimal conditions; Further comprising:

2. The virus detection method of claim 1, wherein in the step of performing the amplification reaction on the specimen sample supplied to the solid phase carrier based on the plurality of amplification reaction conditions, the amplification reaction is performed by using the fixed optimal conditions.

4. The step of setting a plurality of amplification reaction conditions includes: Optimizing the amplification reaction conditions that provide the highest sensitivity while fixing the other multiple amplification reaction conditions after optimization; After optimization, the amplification reaction conditions with the highest sensitivity are fixed, and then the other amplification reaction conditions are subsequently optimized; and setting the amplification reaction conditions with the highest sensitivity after optimization and the other multiple amplification reaction conditions after subsequent optimization as the fixed optimal conditions; The virus detection method according to claim 3, further comprising:

5. The virus detection method of claim 1, further comprising the analysis of a nucleic acid purification reaction, a reverse transcription reaction, a polymerase chain reaction, and an amplification reaction.

6. The virus detection method according to claim 1 , wherein the viruses include human RNA viruses, human DNA viruses, bovine DNA viruses, and porcine DNA viruses.

7. The human RNA viruses include human immunodeficiency virus type 1 (HIV-I), human immunodeficiency virus type 2 (HIV-II), hepatitis A virus (HAV), hepatitis C virus (HCV), human lymphotropic virus type 1 (HTLV-I), and human lymphotropic virus type 2 (HTLV-II).

7. The method for detecting a virus according to claim 6, wherein the virus comprises a virus selected from the group consisting of HTLV-1, HTLV-2, and HTLV-II.

8. The human DNA viruses include Hepatitis B Virus (HBV), B19 Parvovirus (Parvovirus B19), Epstein-Barr Virus Type 4 (EBV), Human Cytomegalovirus (HCMV), Human Adenovirus (HAdV), Human Herpesvirus Type 6 (HHV-6), Human Herpesvirus Type 7 (HHV-7), and Human Herpesvirus Type 8 (HHV-8). 8, HHV-8), Human Papillomavirus Type 16 (HPV-16), Human Papillomavirus Type 18 (HPV-18), BK virus, and JC virus.

9. 7. The virus detection method according to claim 6, wherein the bovine DNA virus includes bovine polyomavirus (BPyV).

10. The virus detection method according to claim 6, wherein the porcine DNA viruses include porcine circovirus type 1 (PCV-1), porcine circovirus type 2 (PCV-2), and porcine parvovirus (PPV).

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