Nucleic acid combination for detecting acute hemorrhagic conjunctivitis pathogen, kit comprising same and application thereof

Abstract

The invention discloses a nucleic acid composition for detecting acute hemorrhagic conjunctivitis pathogens, a kit containing the nucleic acid composition and application of the nucleic acid composition. The nucleic acid combination comprises one or more of the following groups: a primer probe group 1: upstream and downstream primers and probes for detecting the enterovirus 70, wherein the sequence of the primer probe group 1 is shown as SEQ ID NO: 1-3; a primer probe group 2: upstream and downstream primers and probes for detecting 24-type variants of the coxsackie virus group A, wherein the sequences of the upstream and downstream primers and probes are shown as SEQ ID NO: 4-6; a primer probe group 3: upstream and downstream primers and probes for detecting the adenovirus subgenus B, wherein the sequences of the upstream and downstream primers and probes are shown as SEQ ID NO: 7-9; the sequence of the primer probe group 4 is shown as SEQ ID NO: 10 to SEQ ID NO: 12, and the primer probe group 4 is used for detecting upstream and downstream primers and probes of the adenovirus subgenus D. When the nucleic acid combination is used for detection, at least the advantages of high detection cost, high detection efficiency, high sensitivity and high specificity are achieved.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a nucleic acid combination for detecting pathogens of acute hemorrhagic conjunctivitis, a kit containing the same, and uses thereof. Background Art

[0002] Acute hemorrhagic conjunctivitis (AHC) is a Class C infectious disease among the legally reportable infectious diseases. It is mainly transmitted through contact and is very likely to cause clustered epidemics. The disease also has the characteristics of a short incubation period, rapid onset, severe eye irritation symptoms, conjunctival congestion, strong infectiousness, and easy transmission. It is prevalent worldwide.

[0003] In the summer and autumn, if a large number of patients with acute conjunctivitis appear in one area or unit, or if a large number of patients with acute conjunctivitis with a very short incubation period, rapid onset, and rapid contact transmission suddenly appear in hospital outpatient clinics or medical rooms, we must be highly vigilant against the spread of acute hemorrhagic conjunctivitis.

[0004] The diagnosis of acute hemorrhagic conjunctivitis requires a clinical diagnosis of AHC based on the medical history, contact history, epidemiological history combined with clinical symptoms and signs. The exact diagnosis of acute hemorrhagic conjunctivitis must be confirmed by laboratory etiology: (i) EV70 or CA24v is isolated by conjunctival swab or conjunctival scraping culture. (ii) Viral antigen is detected by indirect immunofluorescence technique or enzyme-linked immunosorbent assay of conjunctival scraping. (iii) Biphasic serological examination. The titer of anti-EV70 or anti-CA24v antibodies in the patient's recovery serum is 4 times or more higher than that in the acute phase. (iv) EV70 is detected in conjunctival specimens by reverse transcription polymerase chain reaction. Clinical diagnosis plus any positive laboratory pathogen test is considered a confirmed case. The existing nucleic acid diagnostic method for acute hemorrhagic conjunctivitis is based only on nucleic acid detection of the two main pathogens, EV70 and CA24V.

[0005] Therefore, it is urgent to establish a method that can simultaneously detect multiple pathogens of acute hemorrhagic conjunctivitis. Summary of the invention

[0006] Based on this, it is necessary to provide at least a nucleic acid combination for detecting pathogens of acute hemorrhagic conjunctivitis, a kit containing the same, and its use.

[0007] In a first aspect of the present application, a nucleic acid combination for detecting pathogens of acute hemorrhagic conjunctivitis is provided, the nucleic acid combination comprising one or more groups selected from the following groups: primer probe group 1: upstream and downstream primers and probes for detecting enterovirus 70 (EV70) with sequences as shown in SEQ ID NOs: 1 to 3; primer probe group 2: upstream and downstream primers and probes for detecting coxsackievirus group A type 24 variant (CA24v) with sequences as shown in SEQ ID NOs: 4 to 6; primer probe group 3: upstream and downstream primers and probes for detecting adenovirus B subgenus (ADV B) with sequences as shown in SEQ ID NOs: 7 to 9; and primer probe group 4: upstream and downstream primers and probes for detecting adenovirus D subgenus (ADV D) with sequences as shown in SEQ ID NOs: 10 to 12.

[0008] In a second aspect of the present application, a kit is provided, comprising the nucleic acid combination as described in the first aspect.

[0009] In the third aspect of the present application, a method for detecting pathogens of acute hemorrhagic conjunctivitis is provided, comprising the following steps: extracting nucleic acid from a sample to be tested to obtain a nucleic acid sample; performing PCR amplification on the nucleic acid sample using the kit described in the second aspect; and obtaining and analyzing the results.

[0010] This application provides an illustrative method for efficiently, quickly and at low cost to identify the pathogen of acute hemorrhagic conjunctivitis. The fluorescent quantitative PCR method can be used to extract nucleic acid from patient samples, and amplify enterovirus 70 (EV70), Coxsackie A24 variant (CA24V) and adenovirus conservative regions to achieve the purpose of rapid detection and identification of pathogens causing acute hemorrhagic conjunctivitis. The advantages of this method are at least that the detection cost and detection efficiency are high while taking into account sensitivity and specificity, which is of great significance for the epidemiological detection of acute hemorrhagic conjunctivitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0012] Figure 1 This is the amplification result of the first set of primer-probe single detection system of CA24V in one embodiment of the present application.

[0013] Figure 2 This is the amplification result of the first set of primer-probe combined detection system for CA24V in one embodiment of the present application.

[0014] Figure 3 This is the EV70 test result in one embodiment of the present application - positive.

[0015] Figure 4 In one embodiment of the present application, the CA24V test result is positive.

[0016] Figure 5 It is the ADV test result-positive in one embodiment of the present application.

[0017] Figure 6 The detection result of the housekeeping gene GAPDH in one embodiment of the present application is positive.

[0018] Figure 7 It is a positive control in one embodiment of the present application.

[0019] Figure 8 This is the EV70 sensitivity detection result in one embodiment of the present application.

[0020] Fig. 9 This is the CA24V sensitivity detection result in one embodiment of the present application.

[0021] Fig.10 This is the adenovirus sensitivity test result in one embodiment of the present application.

[0022] Fig.11 This is the EV70 precision result in one embodiment of the present application.

[0023] Fig.12 This is the CA24V precision result in one embodiment of the present application.

[0024] Fig.13 This is the ADV precision result in one embodiment of the present application.

[0025] Fig.14 This is a specific detection result in one embodiment of the present application. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0028] In this application, unless otherwise specified, "one or more groups" means any group of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0029] The terms "combination", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0030] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0031] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0032] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0033] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0034] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0035] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0036] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0037] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0038] The first aspect of the present application provides a nucleic acid combination for detecting pathogens of acute hemorrhagic conjunctivitis, wherein the nucleic acid combination comprises one or more groups selected from the group consisting of:

[0039] Primer probe set 1: The sequences are shown in SEQ ID NOs: 1 to 3, which are upstream and downstream primers and probes for detecting enterovirus 70 (EV70);

[0040] Primer probe set 2: the sequences are shown in SEQ ID NOs: 4 to 6, upstream and downstream primers and probes for detecting Coxsackievirus group A type 24 variant (CA24v);

[0041] Primer probe set 3: upstream and downstream primers and probes for detecting adenovirus subgenus B (ADV B) with sequences as shown in SEQ ID NOs: 7 to 9; and,

[0042] Primer probe set 4: The sequences are shown in SEQ ID NOs: 10 to 12, which are upstream and downstream primers and probes for detecting adenovirus subgenus D (ADV D).

[0043] In some embodiments, the nucleic acid combination includes primer probe set 1, primer probe set 2, primer probe set 3, or primer probe set 4.

[0044] In some embodiments, the nucleic acid combination includes any two of primer probe set 1, primer probe set 2, primer probe set 3, and primer probe set 4.

[0045] In some embodiments, the nucleic acid combination includes primer probe set 1 and primer probe set 2 at the same time.

[0046] The nucleic acid combination can be used to simultaneously detect EV70 and CA24v in a sample.

[0047] In some embodiments, the nucleic acid combination includes primer probe set 1 and primer probe set 3 at the same time.

[0048] The nucleic acid combination can be used to simultaneously detect EV70 and ADV B in a sample.

[0049] In some embodiments, the nucleic acid combination includes primer probe set 1 and primer probe set 4 at the same time.

[0050] The nucleic acid combination can be used to simultaneously detect EV70 and ADV D in a sample.

[0051] In some embodiments, the nucleic acid combination includes primer probe set 2 and primer probe set 3 at the same time.

[0052] The nucleic acid combination can be used to simultaneously detect CA24v and ADV B in a sample.

[0053] In some embodiments, the nucleic acid combination includes primer probe set 2 and primer probe set 4 at the same time.

[0054] The nucleic acid combination can be used to simultaneously detect CA24v and ADV D in a sample.

[0055] In some embodiments, the nucleic acid combination includes any three of primer probe set 1, primer probe set 2, primer probe set 3, and primer probe set 4.

[0056] In some embodiments, the nucleic acid combination includes primer probe set 1, primer probe set 2, and primer probe set 3.

[0057] The nucleic acid combination can be used to simultaneously detect EV70, CA24v and ADV B in a sample.

[0058] In some embodiments, the nucleic acid combination includes primer probe set 2, primer probe set 3, and primer probe set 4.

[0059] The nucleic acid combination can be used to simultaneously detect CA24v, ADV B and ADV D in a sample.

[0060] In some embodiments, the nucleic acid combination includes primer probe set 1, primer probe set 2, and primer probe set 4.

[0061] The nucleic acid combination can be used to simultaneously detect EV70, CA24v and ADV D in a sample.

[0062] In some embodiments, the nucleic acid combination includes primer probe set 1, primer probe set 3, and primer probe set 4.

[0063] The nucleic acid combination can be used to simultaneously detect EV70, ADV B and ADV D in a sample.

[0064] In some embodiments, the nucleic acid combination further includes primer probe set 5: upstream and downstream primers and probes for detecting GAPDH, whose sequences are shown in SEQ ID NOs: 13 to 15.

[0065] The second aspect of the present application provides a kit comprising the nucleic acid combination as described in the first aspect.

[0066] In some embodiments, the kit includes primer probe set 1 to primer probe set 4.

[0067] In some embodiments, the primer probe set 1, the primer probe set 2, and the primer probe set for detecting adenovirus are each labeled with a fluorescent reporter group and are different from each other and do not interfere with each other.

[0068] In some embodiments, the probes in the primer probe set 1 to the primer probe set 4 are each labeled with a fluorescent reporter group and are different from each other and do not interfere with each other.

[0069] The fluorescent reporter group in the present application can be a conventional fluorescent reporter group in the art. For example, the fluorescent reporter group is selected from the group consisting of FAM, HEX, VIC, ROX, CY5 and Quasar 705.

[0070] In some embodiments, the primer probe set 1 is labeled with a fluorescent reporter group FAM, the primer probe set 2 is labeled with a fluorescent reporter group HEX, and the primer probe set for detecting adenovirus is labeled with ROX.

[0071] In some embodiments, the primer probe set 1 is labeled with a fluorescent reporter group FAM, the primer probe set 2 is labeled with a fluorescent reporter group HEX, the primer probe set for detecting adenovirus is labeled with ROX; and the primer probe set 5 is labeled with a fluorescent reporter group CY5.

[0072] In some embodiments, the kit further comprises a positive quality control and / or a negative quality control.

[0073] In some embodiments, the kit further comprises a nucleic acid release reagent, a nucleic acid extraction reagent, a DNA polymerase (such as Taq enzyme), a reverse transcriptase (RT enzyme), a ribonuclease inhibitor, dNTP (U) s, a PCR buffer, and Mg. 2+ At least one of .

[0074] The third aspect of the present application provides a method for detecting pathogens of acute hemorrhagic conjunctivitis, comprising the following steps:

[0075] Extracting nucleic acid from the sample to be tested to obtain a nucleic acid sample;

[0076] Perform PCR amplification on the nucleic acid sample using the kit as described in the second aspect; and,

[0077] Obtain and analyze results.

[0078] In some embodiments, in each reaction system for PCR amplification, the concentration of each primer pair is 100 nM to 500 nM, and the concentration of each probe is 50 nM to 250 nM.

[0079] In some embodiments, in each reaction system for PCR amplification, the concentration of each primer pair is 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, or a range or value between any two values.

[0080] In some embodiments, in each reaction system for PCR amplification, the concentration of each probe is 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, or a range or value between any two values.

[0081] In some embodiments, in each reaction system for PCR amplification, the concentration of the DNA polymerase is 5 U / μL to 15 U / μL, the concentration of the reverse transcriptase is 5 U / μL to 10 U / μL, and the concentration of Mg is Mg. 2+ The concentration of is 4 mM~6 mM, and the concentration of dNTP(U)s is 0.2 mM~0.4 mM.

[0082] In some embodiments, in each reaction system for PCR amplification, the concentration of the DNA polymerase is 5 U / μL, 6 U / μL, 7 U / μL, 8 U / μL, 9 U / μL, 10 U / μL, 11 U / μL, 12 U / μL, 13 U / μL, 14 U / μL, 15 U / μL, or a range or value between any two values.

[0083] In some embodiments, in each reaction system for PCR amplification, the concentration of the reverse transcriptase is 5 U / μL, 6 U / μL, 7 U / μL, 8 U / μL, 9 U / μL, 10 U / μL, or a range or value between any two values.

[0084] In some embodiments, in each reaction system for PCR amplification, Mg 2+ The concentration is 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, or a range or value between any two values.

[0085] In some embodiments, in each reaction system for PCR amplification, the concentration of dNTP(U)s is 0.2 mM, 0.3 mM, 0.4 mM, or a range or value between any two values.

[0086] In some embodiments, the method satisfies one or more of the following conditions 1) to 2):

[0087] 1) The procedure for PCR amplification includes:

[0088] Reverse transcription, 50°C, 4min~5min;

[0089] Pre-denaturation, 95°C, 30 s~1 min;

[0090] Denaturation, 95°C, 10 s~20 s, annealing, 60°C, 16s~20 s, 41-45 cycles; and

[0091] 2) The sample to be tested contains a virus selected from the group consisting of enterovirus 70, Coxsackie A24 variant, and adenovirus types 3, 7, 8, 9, 11, 19, and 37.

[0092] Some examples are provided below.

[0093] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0094] Example 1 Design and application of primers and probes

[0095] BioEdit software was used to compare the homology of the gene sequences of EV70, CA24V, ADV B subgenus (types 3, 7, and 11) and ADV D subgenus (types 8, 9, 19, and 37) retrieved. Beacon Designer 8.14 software was used to design primers and probes in the conserved regions. After retrieval and analysis using the Blast tool in the GenBank database, three sets of primers and probes were designed for each of the target sites of EV70, CA24V, ADVB subgenus (types 3, 7, and 11) and ADV D subgenus (types 8, 9, 19, and 37); they were:

[0096] EV70-F1, EV70-R1 and EV70-P1, EV70-F2, EV70-R2 and EV70-P2, EV70-F3, EV70-R3 and EV70-P3;

[0097] CA24V-F1, CA24V-R1 and CA24V-P1, CA24V-F2, CA24V-R2 and CA24V-P2, CA24V-F3, CA24V-R3 and CA24V-P3;

[0098] ADV B-F1, ADV B-R1 and ADV B-P1, ADV B-F2, ADV B-R2 and ADV B-P2, ADV B-F3, ADVB-R3 and ADV B-P3;

[0099] ADV D-F1, ADV D-R1 and ADV D-P1, ADV D-F2, ADV D-R2 and ADV D-P2, ADV D-F3, ADVD-R3 and ADV D-P3.

[0100] The above primer probes formed 12 sets of single detection systems, which were tested separately with positive samples as templates, and 2 sets of target gene primer probes were screened. The target gene primer probes were EV70-F1, EV70-R1 and EV70-P1, EV70-F3, EV70-R3 and EV70-P3, CA24V-F1, CA24V-R1 and CA24V-P1, CA24V-F2, CA24V-R2 and CA24V-P2, ADV B-F1, ADVB-R1 and ADV B-P1, ADV B-F3, ADV B-R3 and ADV B-P3, ADV D-F2, ADV D-R2 and ADV D-P2, ADV D-F3, ADV D-R3 and ADV B-P3. The two sets of primer probes selected were tested together with the internal standard primer probe to form 16 multiplex systems. The combinations of primer probes are shown in Table 1 below.

[0101] Table 1

[0102]

[0103] Finally, the best system was selected as A7 combination, and finally one joint inspection system was selected, namely:

[0104] EV70-F1, EV70-R1, and EV70-P1; CA24V-F2, CA24V-R2, and CA24V-P2; ADV B-F3, ADV B-R3, and ADV B-P3; ADV D-F2, ADV D-R2, and ADV D-P2; GAPDH-F, GAPDH-R, and GAPDH-P primer-probe combinations.

[0105] During the system screening process, the above-screened single-target gene detection system had a very good detection effect. However, the detection was affected in the combined detection system, and both Ct and fluorescence values ​​performed poorly (see Figure 1 and Figure 2 ). The gene single detection system was adjusted, and the primers and probes used in this application were finally shown in Table 2 below.

[0106] Table 2

[0107]

[0108] Among them: Coxsackie A24 type mutant strains have nucleotide mutations, such as the relatively conservative VP1 gene of CA24V JX196594.1 |Human coxsackievirus A24v Marseille / 2012 / 1 VP1 gene and OR352722.1Coxsackievirus A24 strain CA24v / China.Zhongshan / 08 / 2023 VP1 gene sequence consistency is 93.29%. This application covers new mutant strains while ensuring normal detection of previous strains by designing merged base upstream and downstream primers. The symbol "R" represents adenine (A) or guanine (G), and the symbol "Y" represents thymine (T) or cytosine (C).

[0109] The sample tested was an eye swab. The viral nucleic acid was extracted by magnetic bead method, and the following operations were performed in the sample processing room:

[0110] 1) Take several 1.5 mL centrifuge tubes according to the number of samples to be tested and add 300 μL of sample to each tube.

[0111] 2) Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cover the tube, shake and mix for 30 seconds, and heat at 60℃ for 10 minutes.

[0112] 3) Let stand at room temperature for 1 minute, centrifuge at low speed, place the centrifuge tube on the magnetic separator, and slowly aspirate and discard the waste liquid after 5 minutes (*Be careful not to touch the magnetic beads adsorbed on the inner side of the tube wall).

[0113] 4) Add 200 μL of washing solution 1 and 600 μL of washing solution 2, shake and mix for 30 seconds, centrifuge at low speed, and place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, completely aspirate and discard the liquid.

[0114] 5) Place the centrifuge tube in a centrifuge and centrifuge it at a low speed for a short time. Place the centrifuge tube in a magnetic separator again and magnetically absorb it for 3 minutes to completely absorb the liquid at the bottom of the tube.

[0115] 6) Add 30 μL~100 μL of elution buffer S (80 μL is recommended), shake and mix for 30 seconds to elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, and let it stand at room temperature for 3 minutes; centrifuge at low speed, place the centrifuge tube on the magnetic separator again for 3 minutes, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0116] 7) Pipette 20 μL of the above-treated samples, EV70 / CA24V / ADV-positive control, and EV70 / CA24V / ADV-negative control into the corresponding 0.2 mL PCR reaction tubes, add 30 μL of PCR mixture to each tube, and cover the tubes.

[0117] The real-time fluorescence PCR reaction system was prepared according to Table 3 below.

[0118] Table 3

[0119]

[0120] The PCR amplification program was set according to Table 4.

[0121] Table 4

[0122]

[0123] Result analysis:

[0124] 1) The target detection signal is FAM, HEX (or VIC), ROX, and the human gene internal standard detection signal is CY5;

[0125] 2) Baseline setting: Baseline is generally set to 3-15 cycles, which can be adjusted according to actual conditions. The adjustment principle is: select the area where the fluorescence signal is relatively stable before exponential amplification, avoid the signal fluctuation at the beginning of fluorescence acquisition, and reduce the end point (End) by 1-2 cycles compared to the Ct of the sample with the earliest exponential amplification. Threshold setting: The setting principle is that the threshold line just exceeds the highest point of the normal negative control;

[0126] 3) Interpretation of results

[0127] A) For samples with an amplification curve detected in the FAM channel and Ct≤40, they are reported as EV70 positive; for samples with no amplification curve detected in the FAM channel, or Ct>40, and an amplification curve in the CY5 channel and Ct≤40, they are reported as EV70 negative.

[0128] B) For samples with an amplification curve detected in the HEX channel and Ct≤40, it is reported as CA24V positive; for samples with no amplification curve detected in the HEX channel, or Ct>40, and an amplification curve in the CY5 channel and Ct≤40, it is reported as CA24V negative.

[0129] C) For samples with an amplification curve detected in the ROX channel and Ct≤40, it is reported as ADV positive; for samples with no amplification curve detected in the ROX channel, or Ct>40, and an amplification curve in the CY5 channel and Ct≤40, it is reported as ADV negative.

[0130] D) If no amplification curve (No Ct) value is detected for FAM, ROX, HEX, or CY5 channels, or Ct>40, it means that the cell content of the sample tested is too low or there are interfering substances that inhibit the reaction. The test result of the sample is invalid. The cause should be found and eliminated, and the sample should be resampled and repeated.

[0131] The optimal system screened out was used to detect 1 case of EV70, CA24V, ADV and a positive control, and PCR detection was performed on a Hongshi fluorescent quantitative PCR instrument. The results are as follows Figure 3~Figure 7 shown.

[0132] Sensitivity analysis: Take the gradient dilution plasmid, the concentration is 1000, 500, 200, 100, 50 copies / ml, and test the 5 concentrations respectively, and take 20 μL as template. The results show that the method of this application has high sensitivity and the detection concentration can reach 100 copies / ml ( Figure 8~Figure 10 ).

[0133] Precision analysis: This experiment selected two concentration levels of strong positive and weak positive (20000 copies / ml and 5000 copies / ul respectively) of quality control products for intra-batch precision and inter-batch precision determination, and each sample was repeated 10 times. The results showed that the detection rate of strong positive and weak positive reference products was 100%, and the coefficient of variation (CV) of the Ct value within and between batches was less than 5%. It can be considered that this kit has good detection precision within and between batches ( Figure 11~Figure 13 ).

[0134] Specificity analysis: The system of this application has no cross-reaction with common pathogens of the respiratory tract and other pathogens with similar infection symptoms (such as Bordetella pertussis, respiratory syncytial virus, influenza virus type 1, influenza virus type B, Mycoplasma pneumoniae, Chlamydia pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, Epstein-Barr virus, rhinovirus, human metapneumovirus, Acinetobacter baumannii, Escherichia coli, Serratia marcescens, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, Legionella pneumophila, etc.) ( Fig.14 ).

[0135] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0136] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A nucleic acid combination for detecting pathogens of acute hemorrhagic conjunctivitis, characterized in that: The nucleic acid combination comprises one or more groups selected from the group consisting of: Primer probe set 1: the sequences are shown in SEQ ID NOs: 1 to 3, upstream and downstream primers and probes for detecting enterovirus 70; Primer probe set 2: the sequences are shown in SEQ ID NOs: 4 to 6, upstream and downstream primers and probes for detecting Coxsackievirus group A type 24 variants; Primer probe set 3: upstream and downstream primers and probes for detecting adenovirus subgenus B, with sequences as shown in SEQ ID NOs: 7 to 9; and, Primer probe set 4: The sequences are shown in SEQ ID NOs: 10 to 12, which are upstream and downstream primers and probes for detecting adenovirus subgenus D.

2. The nucleic acid combination according to claim 1, characterized in that It also includes primer probe set 5: upstream and downstream primers and probes for detecting GAPDH, whose sequences are shown in SEQ ID NOs: 13-15.

3. A kit, characterized in that: It comprises the nucleic acid combination according to claim 1 or 2.

4. The kit according to claim 3, characterized in that The primer probe set 1, the primer probe set 2 and the primer probe set for detecting adenovirus are each labeled with a fluorescent reporter group and are different from each other and do not interfere with each other; Optionally, the probes in the primer probe set 1 to the primer probe set 4 are each labeled with a fluorescent reporter group and are different from each other and do not interfere with each other.

5. The kit according to claim 4, characterized in that The fluorescent reporter group is selected from the group consisting of FAM, HEX, VIC, ROX, CY5 and Quasar 705.

6. The kit according to any one of claims 3 to 5, characterized in that The kit further comprises a positive quality control product and / or a negative quality control product; And / or, the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, a DNA polymerase, a reverse transcriptase, a ribonuclease inhibitor, dNTP(U)s, a PCR buffer and Mg 2+ At least one of .

7. A method for detecting pathogens of acute hemorrhagic conjunctivitis, characterized in that: The steps include: Extracting nucleic acid from the sample to be tested to obtain a nucleic acid sample; Performing PCR amplification on the nucleic acid sample using the kit according to any one of claims 3 to 6; and, Obtain and analyze results.

8. The method according to claim 7, characterized in that In each reaction system for PCR amplification, the concentration of each primer pair is 100 nM to 500 nM, and the concentration of each probe is 50 nM to 250 nM.

9. The method according to claim 7 or 8, characterized in that In each reaction system for PCR amplification, the concentration of the DNA polymerase is 5 U / μL~15 U / μL, the concentration of the reverse transcriptase is 5 U / μL~10 U / μL, and the concentration of Mg is 2+ The concentration of is 4 mM~6 mM, and the concentration of dNTP(U)s is 0.2 mM~0.4 mM.

10. The method according to claim 9, characterized in that The method satisfies one or more of the following conditions 1) to 2): 1) The procedure for PCR amplification includes: Reverse transcription, 50°C, 4min~5min; Pre-denaturation, 95°C, 30 s~1 min; Denaturation, 95°C, 10 s-20 s, annealing, 60°C, 16 s-20 s, 41-45 cycles; and 2) The sample to be tested contains a virus selected from the group consisting of enterovirus 70, Coxsackie A24 variant, and adenovirus types 3, 7, 8, 9, 11, 19, and 37.

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