Nucleic acid detection kit for adenovirus, rhinovirus and mycoplasma pneumoniae

Abstract

The invention discloses a nucleic acid detection kit for adenovirus, rhinovirus and mycoplasma pneumoniae, and belongs to the field of molecular biological detection. The probe or the detection kit disclosed by the invention comprises one or more of the following primer combinations: a combination I, which comprises primers with sequences of SEQ ID NO.1-3; the combination II comprises primers of which the sequences are SEQ ID NO.4-7; the combination III comprises primers of which the sequences are SEQ ID NO.8-10; and the fourth combination comprises the following steps: replacing the 13th site in the sequence SEQ ID NO.5 in the second combination with G, and / or replacing the 18th site in the sequence SEQ ID NO.7 with T. The lyophilized reagent disclosed by the invention can be used for simultaneously detecting the pulmonary adenovirus, the rhinovirus and the mycoplasma pneumoniae, so that the detection efficiency of respiratory pathogens is improved; the kit is simple in operation, short in time consumption and high in sensitivity, the lowest detection limit of adenovirus, rhinovirus and mycoplasma pneumoniae is 200 copies / mL, the reagent specificity is high, and the detection of viruses with related and similar symptoms is negative.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology detection, and particularly to a nucleic acid detection kit for adenovirus, rhinovirus and Mycoplasma pneumoniae. Background Art

[0002] Respiratory tract infections rank first in the incidence of acute infectious diseases. Its clinical manifestations are symptoms such as fever, swelling pain, cough, headache, etc. In severe cases, it can cause tracheitis, bronchitis and pneumonia, etc. Respiratory tract infections are most likely to occur in autumn, winter and spring, and are most easily infected in children or the elderly with low immunity. It is mostly transmitted by droplets and is highly contagious. Respiratory tract infections are prone to break out and spread in closed, crowded and humid environments.

[0003] Adenovirus (Adv) belongs to the family Adenoviridae. According to its immunological and biochemical characteristics, adenovirus can be divided into seven subspecies from A to G. It is an important viral pathogen causing respiratory tract infections and can cause severe lower respiratory tract infections in children. Its infection can cause a variety of diseases, including pneumonia, bronchitis, conjunctivitis, gastrointestinal diseases and encephalitis, etc. It is one of the common pathogens of human respiratory tract infections.

[0004] Human rhinovirus (HRV) belongs to the genus Enterovirus of the family Picornaviridae. This virus is one of the most common pathogens causing the common cold. In infants and children, it can cause upper respiratory tract infections, bronchitis, bronchopneumonia, acute asthma and other diseases.

[0005] Mycoplasma pneumoniae (MP) is a common pathogen causing acute respiratory tract infections in adolescents and is one of the smallest microorganisms that can survive independently. Mycoplasma pneumoniae infection can cause mycoplasma pneumonia, which is mainly transmitted by droplets. The incubation period is 2-3 weeks, and the incidence rate is the highest in adolescents. The clinical symptoms are mild. Most patients show general respiratory symptoms such as headache, sore throat, fever, cough, etc.; a few will show critical symptoms such as respiratory failure and multiple organ dysfunction.

[0006] At present, the laboratory detection of respiratory pathogens includes virus isolation and identification, immunofluorescence method, serological detection, multiplex fluorescent PCR method, etc. Among them, the process of virus culture is cumbersome and the positive rate is low; the sensitivity and specificity of immunological methods are poor; the types of serological identification are single, the identification takes a long time and the false positive rate is relatively high, all of which are not suitable for the simultaneous detection of multiple pathogens in a large range, quickly and accurately. The multiplex fluorescent PCR method can simultaneously detect multiple pathogens in a single reaction system. However, due to the mutual interference of different primers or probes contained in the multiplex fluorescent PCR reaction system, the existing products for simultaneously detecting multiple respiratory infection pathogens mostly have deficiencies such as low detection specificity, low detection sensitivity or poor repeatability. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a nucleic acid detection kit for adenovirus, rhinovirus and Mycoplasma pneumoniae.

[0008] The object of the present invention can be achieved by the following technical solutions:

[0009] In a first aspect of the present invention, it relates to a detection probe, comprising one or more of the following primer combinations:

[0010] Combination 1, comprising primers with sequences SEQ ID NO.1-3;

[0011] Combination 2, comprising primers with sequences SEQ ID NO.4-7;

[0012] Combination 3, comprising primers with sequences SEQ ID NO.8-10;

[0013] The sequence SEQ ID NO.5 contains degenerate bases R and S; the SEQ ID NO.7 contains degenerate base Y, where R represents base A or G, S represents base G or C, and Y represents base C or T.

[0014] Optionally, it further comprises a primer combination for detecting the internal standard RNase P, comprising primers with sequences SEQ ID NO.11-13.

[0015] Optionally, the 5' end of the probe is labeled with a fluorescent group and the 3' end is labeled with a quenching group.

[0016] Optionally, the fluorescent group includes VIC, FAM, ROX or CY5.

[0017] Optionally, the quenching group includes MGB, BHQ1 or BHQ2.

[0018] Optionally, the 5' end of SEQ ID NO.3 is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group MGB; the 5' end of SEQ ID NO.7 is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1; the 5' end of SEQ ID NO.10 is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; the 5' end of SEQ ID NO.13 is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ2.

[0019] A second aspect of the present invention relates to a virus detection kit, comprising:

[0020] The above-mentioned detection probe;

[0021] qPCR reaction buffer, enzyme mixture and cryoprotectant

[0022] Optionally, the qPCR reaction buffer contains Tris-HCl, MgCl2, KCl and dNTP; the enzyme mixture includes polymerase and reverse transcriptase; the cryoprotectant includes 3-10% (w / v) trehalose, 1-5% (w / v) PEG, 0.5-2% (w / v) threonine and 1-2% (w / v) BSA.

[0023] A third aspect of the present invention relates to a method for detecting a virus for non-diagnostic purposes, comprising the following steps:

[0024] Prepare a virus detection kit according to the above components;

[0025] Put the virus detection kit and the sample to be tested into a PCR freeze-dried bead reaction tube, shake and mix well, centrifuge instantaneously, and perform a PCR amplification reaction; the PCR reaction steps include: 55°C, 5 min, 95°C, 30 sec, 1 cycle; 95°C, 3 sec, 60°C, 8 sec, 42 cycles.

[0026] Among them, the non-diagnostic purposes include the detection of whether there are pathogens in non-biological samples, such as the detection of whether food samples, or daily necessities and medical utensils are contaminated with adenovirus, rhinovirus and Mycoplasma pneumoniae.

[0027] A fourth aspect of the present invention relates to the application of the above-mentioned detection probe or virus detection kit in the preparation of diagnostic products for adenovirus, rhinovirus and Mycoplasma pneumoniae.

[0028] Further, the dosage of the detection primer in the composition is 0.1 - 0.5 μM; the dosage of the detection probe in the composition is 0.05 - 0.3 μM; the dosage of the internal standard primer in the composition is 0.1 - 0.2 μM; the dosage of the internal standard probe in the composition is 0.05 - 0.10 μM. Each component in each nucleic acid composition of the composition of the present invention exists in a mixed form.

[0029] In one embodiment, the kit includes the primer-probe combination, qPCR reaction buffer, enzyme mixture and lyoprotectant; different from the traditional qPCR reaction kit, it is convenient for reagent storage, transportation and application methods.

[0030] Furthermore, the present kit contains positive control and negative control, which need to be processed synchronously with the specimen to be tested. The positive control consists of pseudoviruses containing specific nucleic acid sequences of adenovirus, rhinovirus, Mycoplasma pneumoniae and internal standard; the negative control is physiological saline.

[0031] Further, the above detection method specifically may include the following steps:

[0032] 1. Sample processing

[0033] 1) Nucleic acid extraction from the sample: Take 200 μL each of the sample to be tested, positive control product, and negative control product for nucleic acid extraction, and operate according to the extraction reagent instruction manual. Among them, the positive control product (lyophilized sphere) needs to be dissolved in 200 μL of reconstitution solution in advance, and shaken and mixed well for standby;

[0034] 2) Sample addition: Take 50 μL each of the above-mentioned processed nucleic acids and add them into the PCR lyophilized sphere reaction tubes respectively. Tighten the tube caps, shake and mix well to ensure that the lyophilized spheres are completely dissolved, and centrifuge instantaneously to spin all the liquid on the tube wall to the bottom of the tube. Immediately start the PCR amplification reaction after covering the tube caps.

[0035] 2. Reagent preparation: The PCR detection reagent of this product is in the form of lyophilized spheres and does not need to be prepared additionally. Prepare the corresponding number of PCR lyophilized spheres according to the number of samples to be tested (including positive control product and negative control product);

[0036] 3. Reaction steps: 55 °C, 5 min, 95 °C, 30 sec, 1 cycle; 95 °C, 3 sec, 60 °C, 8 sec, 42 cycles.

[0037] 4. Result interpretation:

[0038]

[0039] Advantages of the present invention:

[0040] 1. The present invention provides a multiplex qPCR kit for simultaneously detecting adenovirus, rhinovirus, and Mycoplasma pneumoniae, which has the advantages of good detection specificity, high sensitivity, good repeatability, and being free from interference by interfering substances.

[0041] 2. The multiplex qPCR kit provided by the present invention is simple to operate, time-consuming, and can detect the above three common pathogens of respiratory tract infections at one time through a one-tube reaction system. Moreover, the detection results are accurate, suitable for the rapid clinical detection of respiratory tract pathogens, and helpful for timely and correct treatment of the infected pathogens. Brief Description of the Drawings

[0042] The present invention will be further described below in conjunction with the drawings.

[0043] Figure 1 It is the PCR reaction curve graph of rhinovirus for multiple groups of samples in the scheme of the present invention and the liquid scheme without lyoprotectant.

[0044] Figure 2 It is the PCR reaction curve graph of adenovirus for multiple groups of samples in the scheme of the present invention and the liquid scheme without lyoprotectant.

[0045] Figure 3 It is the PCR reaction curve graph of Mycoplasma pneumoniae for multiple groups of samples in the scheme of the present invention and the liquid scheme without lyoprotectant.

[0046] Figure 4 It is the PCR reaction curve graph of detecting rhinovirus at 200 copies / mL during the lowest detection limit analysis.

[0047] Figure 5 It is the PCR reaction curve graph of detecting adenovirus at 200 copies / mL during the lowest detection limit analysis.

[0048] Figure 6 It is the PCR reaction curve graph of detecting Mycoplasma pneumoniae at 200 copies / mL during the lowest detection limit analysis.

[0049] Figure 7 It is the PCR reaction curve graph of detecting rhinovirus at 1000 copies / mL during the precision analysis.

[0050] Figure 8 It is the PCR reaction curve graph of detecting adenovirus at 1000 copies / mL during the precision analysis.

[0051] Figure 9 It is the PCR reaction curve graph of detecting Mycoplasma pneumoniae at 1000 copies / mL during the precision analysis.

[0052] Figure 10It is the PCR reaction curve graph of the primer-probe combination of the present invention during the specific analysis of multiple groups of samples.

[0053] Figure 11 It is the PCR reaction curve graph of different primer combinations of rhinovirus amplifying templates of different types. Detailed implementation manners

[0054] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0055] Example 1 Design of multiplex qPCR primers and probes and configuration of freeze-dried system

[0056] 1. Design and determination of multiplex qPCR primers and probes

[0057] By comparing the sequences of adenovirus (ADV), rhinovirus (HRV) and Mycoplasma pneumoniae (MP), through processes such as designing primers, experimental verification and screening, adjustment and re-verification, the present invention obtained a set of multiplex qPCR primers and probes that can be used to simultaneously detect adenovirus, rhinovirus and Mycoplasma pneumoniae. The primer and probe sequences are shown in Table 1, and the corresponding detection method was established. In addition, the present invention selected the human gene RNase P (ribonuclease P) as an internal standard, and designed the corresponding qPCR primers and probes based on this gene to monitor the sample collection and extraction process and prevent false negatives caused by failed sample nucleic acid extraction.

[0058] Table 1 Multiplex qPCR primer and probe sequences

[0059]

[0060] The present invention realized the detection of different types of rhinovirus (rhinovirus types are A, B and C) by designing one more primer, while ensuring the detection specificity and sensitivity of the primers and probes and the accuracy of the detection.

[0061] Those skilled in the art should understand that in some other embodiments of the present invention, without affecting the detection function, the sequences SEQ ID NO.5 or 7 can use degenerate bases. Specifically, R can be used to represent base A or G, S can be used to represent base G or C, and Y can be used to represent base C or T. Preferably, in some specific embodiments, R takes base A and S takes base G.

[0062] In this embodiment, the 5' end of the probe for detecting adenovirus is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group MGB; the 5' end of the probe for detecting rhinovirus is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1; the 5' end of the probe for detecting Chlamydia pneumoniae is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2. The 5' end of the probe for detecting the internal standard is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ2.

[0063] 1. Configuration of the freeze-dried system

[0064] Configure the reaction system according to Table 2

[0065] The reaction system described in Table 2

[0066] Component Per reaction (μL) qPCR reaction buffer 10 Enzyme mixture 2.5 SEQ ID NO:1 (100 μM) 0.15 SEQ ID NO:2 (100 μM) 0.15 SEQ ID NO:3 (100 μM) 0.05 SEQ ID NO:4 (100 μM) 0.25 SEQ ID NO:5 (100 μM) 0.25 SEQ ID NO:6 (100 μM) 0.25 SEQ ID NO:7 (100 μM) 0.15 SEQ ID NO:8 (100 μM) 0.05 SEQ ID NO:9 (100 μM) 0.05 SEQ ID NO:10 (100 μM) 0.04 SEQ ID NO:11 (100 μM) 0.05 SEQ ID NO:12 (100 μM) 0.05 SEQ ID NO:13 (100 μM) 0.025 DEPC water 0.985 Lyoprotectant 15 Total amount 30

[0067] The qPCR reaction buffer contains components such as Tris-HCl, MgCl2, KCl, and dNTP.

[0068] The enzyme mixture contains components such as polymerase and reverse transcriptase.

[0069] The lyoprotectant contains components such as trehalose, PEG, threonine, and BSA.

[0070] Preparation of PCR freeze-dried beads: Referring to the reaction system in Table 2, drop 30 μL per drop into a vial filled with liquid nitrogen, quickly transfer it to a freeze dryer and perform freeze-drying according to the freeze-drying procedure in Table 3. After freeze-drying, freeze-dried beads can be obtained, and the freeze-dried beads are aliquoted into an eight-well strip.

[0071] Table 3 Freeze-drying procedure:

[0072]

[0073] Establishment of the detection method in Example 2

[0074] For the multiplex qPCR primers and probes shown in Table 1, the present invention constructs corresponding detection methods using inactivated cultures of adenovirus (types 1, 2, 3, 4, 5, 7, 55), rhinovirus (types A, B, and C), and Mycoplasma pneumoniae, as well as negative clinical samples for simultaneous detection of adenovirus, rhinovirus, and Mycoplasma pneumoniae; mix the primers and probes shown in Table 1 into one tube, perform bead dropping and freeze-drying, and obtain PCR freeze-dried beads containing primers and probes, and simultaneously detect the above three common pathogens of respiratory tract infections through one PCR freeze-dried bead.

[0075] The reaction system is a PCR dry pellet, and the nucleic acid of the sample to be tested is 50 μL. The PCR dry pellet contains multiplex qPCR primers and probes for detecting adenovirus, rhinovirus, and Mycoplasma pneumoniae, polymerase, reverse transcriptase, components such as Tris-HCl, MgCl2, KCl, and dNTP.

[0076] The reaction procedure is as follows: 55°C for 5 min, 95°C for 30 sec, 1 cycle; 95°C for 3 sec, 60°C for 8 sec (collect fluorescence), 42 cycles.

[0077] The present invention also optimizes the reaction procedure. Under the condition that the cycling temperature and time remain unchanged, the reverse transcription temperatures of 55°C and 50°C and the reverse transcription times of 3 min, 5 min, and 10 min are compared; under the condition that the reverse transcription temperature and time remain unchanged, the denaturation times of 5 sec, 3 sec, and 2 sec at 95°C and the extension times of 30 sec, 15 sec, 8 sec, and 5 sec at 60°C are compared. Under the condition that the reverse transcription and cycling temperatures and times remain unchanged, the cycle numbers of 40, 42, and 45 are compared. To find an amplification procedure with equivalent performance and the shortest amplification time.

[0078] The results show that in the constructed reaction system, the amplification procedure of 55°C for 5 min, 95°C for 30 sec, 1 cycle; 95°C for 3 sec, 60°C for 8 sec (collect fluorescence), 42 cycles has no obvious change in performance and the shortest amplification time.

[0079] Example 3 Nucleic Acid Detection Kit for Detecting Adenovirus, Rhinovirus, and Mycoplasma pneumoniae

[0080] Based on the multiplex qPCR primers, probes, and detection methods described in Example 1 and Example 2, the present invention also provides a multiplex qPCR kit that can be used to simultaneously detect adenovirus (ADV), rhinovirus (HRV), and Mycoplasma pneumoniae (MP).

[0081] 1. Composition of the Kit

[0082] The nucleic acid detection kit for detecting adenovirus, rhinovirus, and Mycoplasma pneumoniae includes a PCR dry pellet, a positive control, a negative control, and a reconstitution solution.

[0083] The PCR dry pellet contains multiplex qPCR primers and probes for detecting adenovirus, rhinovirus, and Mycoplasma pneumoniae, polymerase, reverse transcriptase, components such as Tris-HCl, MgCl2, KCl, and dNTP.

[0084] The positive control consists of pseudoviruses containing specific nucleic acid sequences of adenovirus, rhinovirus, Mycoplasma pneumoniae, and an internal standard.

[0085] The negative control is physiological saline.

[0086] The reconstitution solution is DEPC water.

[0087] 2. Method of using the kit

[0088] The present invention also provides a method of using the kit. The applicable sample type is oropharyngeal swab. The specific steps are as follows:

[0089] 2.1 Nucleic acid extraction: Take 200 μL each of the sample to be tested, positive control product, and negative control product for nucleic acid extraction. Among them, the positive control product (lyophilized bead) needs to be dissolved in advance with 200 μL of reconstitution solution and shaken well for later use. In the embodiment of the present invention, the reagent used for extracting sample nucleic acid is the nucleic acid extraction kit (magnetic bead method) (registration number: Su Xu Medical Equipment Preparation 20210027) produced by Jiangsu Xunrui Biotechnology Co., Ltd.;

[0090] 2.2 Sample addition: Take 50 μL of the above-treated nucleic acid and add it to each of the octuplets containing PCR lyophilized beads. Tighten the tube caps, shake well to ensure that the PCR lyophilized beads are completely dissolved, and centrifuge instantaneously to spin all the liquid on the tube wall to the bottom of the tube, and perform an amplification reaction in a real-time fluorescence PCR instrument; The reaction program is 55°C, 5 min, 95°C, 30 sec, 1 cycle; 95°C, 3 sec, 60°C, 8 sec (collect fluorescence), 42 cycles;

[0091] 2.3 Result interpretation: Fluorescence channels are sequentially selected as FAM, VIC, ROX, and Cy5. After qPCR, judge the negativity and positivity of the corresponding pathogen nucleic acid through the curves and Ct values of different fluorescence channels

[0092] When the Ct value corresponding to each channel ≤ the positive judgment value of 40 and the amplification curve has an obvious exponential growth, the detection result of the corresponding channel is judged as positive; when the Ct value > the positive judgment value of 40 or there is no Ct value, the corresponding channel is judged as negative; specifically as follows:

[0093]

[0094]

[0095] Note:

[0096] 1. Each experiment needs to detect the positive control product and the negative control product. The detection result can be determined only when the results meet the quality control requirements in the test method.

[0097] 2. For the detection of the target gene being positive, the detection result of the internal standard may be positive, or it may be negative due to the amplification of a high concentration of the target gene.

[0098] 3. Negative result: For the sample to be tested in the detection channel, if the Ct value of the CY5 channel (internal standard) is ≤ 40, but there is no obvious fluorescence increase and no typical S-shaped amplification curve in the FAM channel (HRV), VIC channel (Adv), and ROX channel (MP), or the Ct values of the FAM channel (HRV), VIC channel (Adv), and ROX channel (MP) of the sample to be tested are all greater than their positive judgment values or NoCt or Undet., then the sample is determined to be a negative sample.

[0099] Example 4 Comparison between PCR freeze-dried beads and liquid reagent without freeze-drying protectant

[0100] In the solution of the present invention (PCR freeze-dried beads freeze-dried with a primer-probe qPCR reaction solution and 1:1 freeze-drying protectant) and the liquid solution without freeze-drying protectant (containing a primer-probe qPCR reaction solution) were compared. Under the condition that the final concentration of the input samples was the same, the samples were added.

[0101] Liquid solution without freeze-drying protectant: In the reaction system described in Table 2, the freeze-drying protectant was replaced with enzyme-free water and no freeze-drying was performed.

[0102] The results are shown in Table 4 and Figures 1 - 3 as follows, indicating that the result of the solution of the present invention (PCR freeze-dried beads freeze-dried with a primer-probe qPCR reaction solution and 1:1 freeze-drying protectant) is slightly better than that of the liquid solution without freeze-drying protectant (containing a primer-probe qPCR reaction solution).

[0103] Table 4 Statistical table of Ct value results of different solutions

[0104]

[0105] Note: When the final reaction concentration is the same, the larger the Ct value, the worse the detection effect. NoCt indicates negative.

[0106] Example 5 Minimum detection limit test

[0107] According to the implementation steps in Example 1 and Example 2, experiments were carried out.

[0108] Inactivated cultures or pseudoviruses diluted to 200 copies / mL were used as samples for nucleic acid extraction. Twenty replicate wells were extracted for each sample, and PCR amplification and result judgment were carried out according to the method in Example 3.

[0109] The results are shown in Table 5 and Figures 4 - 6 as follows. The detection rate was 100%, indicating that the minimum detection limit met the requirements (when the number of positive detections in 20 extraction replicate wells ≥ 19, that is, the detection rate ≥ 95%, it can be determined that the minimum detection limit meets the requirements); the minimum detection limits of the kit of the present invention for the detection of adenovirus, rhinovirus, and Mycoplasma pneumoniae were all 200 copies / mL.

[0110] Table 5 Results of the minimum detection limits for detecting adenovirus, rhinovirus, and Mycoplasma pneumoniae in the present invention

[0111]

[0112] Note: The larger the Ct value, the worse the detection effect, and NoCt indicates negative.

[0113] Example 6 Precision test

[0114] Using the inactivated culture or pseudovirus diluted to 1000 copies / mL as the sample, nucleic acid extraction was performed. 10 replicate wells were extracted for each sample, and PCR amplification and result judgment were carried out according to the method in Example 3.

[0115] The results are shown in Table 6 and Figures 7 - 9 as follows. The CV values of the Ct values of the 10 extracted replicate wells were all ≤ 5%, indicating that the precision of the kit for adenovirus, rhinovirus, and Mycoplasma pneumoniae in the present invention meets the requirements.

[0116] Table 6 Precision results for detecting adenovirus, rhinovirus, and Mycoplasma pneumoniae in the present invention

[0117]

[0118]

[0119] Note: The larger the Ct value, the worse the detection effect, and NoCt indicates negative.

[0120] Example 7 Specificity test

[0121] The primer-probe combination of the present invention was used to detect the remaining common respiratory pathogens, such as parainfluenza virus types 1, 2, 3, and 4, influenza A virus subtypes H1N1, H1N1-2009, H3N2, H5N1, H7N9, influenza B virus lineages Yamagata and Victoria, respiratory syncytial virus types A and B, metapneumovirus types A and B, coronavirus types 229E, OC43, NL63, and HKU1, novel coronavirus, enterovirus 71, coxsackievirus A16, bocavirus, cytomegalovirus, herpes simplex virus type I, varicella-zoster virus, Epstein-Barr virus, Bordetella pertussis, Chlamydia pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Lactobacillus crispatus, Legionella pneumophila, Moraxella catarrhalis, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Mycobacterium tuberculosis, Streptococcus salivarius, Pneumocystis, Candida albicans, Klebsiella pneumoniae (the virus at

[0122] ≥10 5 copies / mL, bacteria at ≥10 6There was no cross - reaction (CFU / mL), and the experimental results were as Figure 10 shown.

[0123] In the present invention, adenovirus, rhinovirus, and Mycoplasma pneumoniae are jointly detected, and there is mutual interference between the primers and probes. Using primer design software, several sets of qPCR primers and probes were designed for the target nucleic acid sequences of adenovirus, rhinovirus, and Mycoplasma pneumoniae respectively. However, through single - channel test detection, it was found that the sensitivity or specificity of individual primer pairs did not meet the requirements.

[0124] For example, in Comparative Example 1, the upstream primer HRV - F1 for detecting rhinovirus designed in the present invention was changed to HRV - F2, and in Comparative Example 2, the upstream HRV - 1 - F2 primer was changed to HRV - F3. Their sequences are as follows:

[0125] Rhinovirus upstream primer HRV - F2: GGTGHGAAGAGCCSCGTGTG

[0126] Rhinovirus upstream primer HRV - F3: GGGTGTGAAGAGTCTAHTGTGCT

[0127] From the detection results in Table 7 and Figure 11 it can be seen that among the 10 positive samples, the present invention's scheme can detect 8 positive samples of rhinovirus types A, B, and C, and samples of Coxsackievirus A16 and Enterovirus 71 were not detected; in Comparative Example 1 above, when the upstream was changed to HRV - F2, samples of rhinovirus types A and B could be detected, and samples of rhinovirus type C, Coxsackievirus A16, and Enterovirus 71 were not detected; in Comparative Example 2 above, when the upstream HRV - 1 - F2 was changed to HRV - F3, all 10 positive samples could be detected, but there was non - specificity.

[0128] Table 7 Ct value inspection table for different primer combinations of rhinovirus amplifying different types of templates

[0129]

[0130] Note: The larger the Ct value, the worse the detection effect, and NoCt indicates negative.

[0131] From the above experimental data, it can be seen that in the detection test of 10 virus samples, the Ct values obtained using the probe combination of the present invention are significantly lower than those of the probe combinations in the comparative examples. Therefore, the probe combination and its kit provided by the present invention have high sensitivity and specificity in the detection of rhinovirus. At the same time, it is also proved that although there are multiple primers in the kit of the present invention, the interference between the primers is small, and it has high commercial application value.

[0132] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0133] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A detection probe, characterized in that: Include one or more of the following primer combinations: Combination 1, comprising: primers having sequences of SEQ ID NOs. 1 to 3; Combination 2, comprising: primers having sequences of SEQ ID NOs. 4 to 7; Combination 3, comprising: primers having sequences of SEQ ID NOs. 8 to 10; The sequence SEQ ID NO.5 contains degenerate bases R and S; the sequence SEQ ID NO.7 contains degenerate base Y, R represents base A or G, S represents base G or C, and Y represents base C or T.

2. The detection probe according to claim 1, characterized in that Also included is a primer combination for detecting internal standard RNase P, including primers with sequences of SEQ ID NOs. 11 to 13.

3. The detection probe according to claim 1, characterized in that The 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

4. The detection probe according to claim 3, characterized in that The fluorescent group includes VIC, FAM, ROX or CY5.

5. The detection probe according to claim 3, characterized in that: The quenching group includes MGB, BHQ1 or BHQ2.

6. The detection probe according to claim 2, characterized in that: The 5' end of SEQ ID NO.3 is labeled with a fluorescent group VIC, and the 3' end is labeled with a quenching group MGB; the 5' end of SEQ ID NO.7 is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1; the 5' end of SEQ ID NO.10 is labeled with a fluorescent group ROX, and the 3' end is labeled with a quenching group BHQ2; the 5' end of SEQ ID NO.13 is labeled with a fluorescent group CY5, and the 3' end is labeled with a quenching group BHQ2.

7. A virus detection kit, characterized in that: include: The detection probe according to any one of claims 1 to 6; qPCR reaction buffer, enzyme mix, and lyoprotectant.

8. The virus detection kit according to claim 7, characterized in that: The qPCR reaction buffer comprises Tris-HCl, MgCl2, KCl and dNTP; the enzyme mixture comprises polymerase and reverse transcriptase; the lyophilized protective agent comprises 3-10% (w / v) trehalose, 1-5% (w / v) PEG, 0.5-2% (w / v) threonine and 1-2% (w / v) BSA.

9. A method for detecting a virus for non-diagnostic purposes, characterized in that: The following steps are involved: A virus detection kit is obtained by configuring the components according to claim 7 or 8; The virus detection kit and the sample to be tested are placed in a PCR freeze-dried ball reaction tube, shaken and mixed, centrifuged instantly, and PCR amplification reaction is performed; The PCR reaction steps include: 55°C, 5 min, 95°C, 30 sec, 1 cycle; 95°C, 3sec, 60°C, 8sec, 42 cycles.

10. Use of the detection probe according to any one of claims 1 to 6 or the virus detection kit according to claim 7 or 8 in the preparation of diagnostic products for adenovirus, rhinovirus and Mycoplasma pneumoniae.

Images