Composition for typing detection of human parainfluenza virus and kit thereof

Abstract

The invention discloses a composition for human parainfluenza virus typing detection and a kit thereof. The composition comprises a primer pair and a probe corresponding to the primer pair, the primer pairs comprise a primer pair 1 aiming at the parainfluenza virus type I, a primer pair 2 aiming at the parainfluenza virus type II and a primer pair 3 aiming at the parainfluenza virus type III; the probes comprise a specific probe 1 aiming at the parainfluenza virus type I, a specific probe 2 aiming at the parainfluenza virus type II and a specific probe 3 aiming at the parainfluenza virus type III. The primer and probe combination provided by the invention has the advantages of high sensitivity, high specificity and highly accurate detection result, and the kit provided by the invention can meet the requirement that the detection limit is not higher than 200copies / mL.

Description

Technical Field

[0001] The invention belongs to the field of molecular biological detection, and in particular relates to a composition and a kit for human parainfluenza virus typing detection. Background Art

[0002] Human parainfluenza virus (HPIV) belongs to the Paramyxoviridae family and is a single-stranded RNA virus. Human parainfluenza virus can be divided into four serotypes, namely type I to type IV. Serotype IV can even be further divided into HPIV-4A and HPIV-4B. The four types of parainfluenza virus have different clinical and epidemiological characteristics. The most typical clinical feature of type I and type II is laryngotracheobronchitis in children. Type I is the main cause of this type of laryngotracheobronchitis in children, while type II is second. Types I and III are one of the main causes of lower respiratory tract infections in infants, young children, adults, immunocompromised people and the elderly. Type IV causes milder symptoms and mostly causes upper respiratory tract infections in adults and children. Currently, the market mainly conducts nucleic acid tests on types I, II and III. At present, the traditional fluorescent quantitative PCR technology is mainly used to detect parainfluenza virus, but the sensitivity is usually not high and the test results are inaccurate. Summary of the invention

[0003] The invention discloses a composition and a kit for human parainfluenza virus typing detection, so as to solve the problems of low sensitivity and inaccurate detection results of traditional detection methods.

[0004] In a first aspect of the present application, a composition for human parainfluenza virus typing detection is provided, comprising: a primer pair and a probe corresponding thereto;

[0005] The primer pairs include: primer pair 1 for parainfluenza virus type I, whose nucleotide sequences are shown in SEQ ID NO.01 and SEQ ID NO.02; primer pair 2 for parainfluenza virus type II, whose nucleotide sequences are shown in SEQ ID NO.03 and SEQ ID NO.04; primer pair 3 for parainfluenza virus type III, whose nucleotide sequences are shown in SEQ ID NO.05 and SEQ ID NO.06;

[0006] The probes include: a specific probe 1 for parainfluenza virus type I, whose nucleotide sequence is shown in SEQ ID NO.09; a specific probe 2 for parainfluenza virus type II, whose nucleotide sequence is shown in SEQ ID NO.10; a specific probe 3 for parainfluenza virus type III, whose nucleotide sequence is shown in SEQ ID NO.11;

[0007] SEQ ID NO.01: 5′-TAGARGCAGCATACACTACTACATCRT-3′;

[0008] SEQ ID NO.02: 5′-GGCTGGTTTGGTTGATTTCA-3′;

[0009] SEQ ID NO.03: 5′-TCCTATGTCATCCGTTATCACTCA-3′;

[0010] SEQ ID NO.04: 5′-CCATCATGACTTGGGAATTGTTAA-3′;

[0011] SEQ ID NO.05: 5′-GTGTCATCTGTCATATTAGACTCACAA-3′;

[0012] SEQ ID NO.06: 5′-GCTGAGAGTGTTCTATTTCGGAT-3′;

[0013] SEQ ID NO.09: 5′-TCACTCAYTTCGGAAAGGGCTACTGC-3′;

[0014] SEQ ID NO.10: 5′-GAGTGCTGTCTYATTTGTCCATTGTCTG-3′;

[0015] SEQ ID NO. 11: 5'-AACCCAGTCATAACTTACTCAACAGCAACC-3π.

[0016] Optionally, the primer pair further comprises: a primer pair 4 for the human β-globin gene used as an internal reference, whose nucleotide sequence is shown in SEQ ID NO.07 and SEQ ID NO.08; the probe further comprises a specific probe 4 for the human β-globin gene used as an internal reference, whose nucleotide sequence is shown in SEQ ID NO.12;

[0017] SEQ ID NO.07: 5′-GCTTACATTTGCTTCTGACA-3′;

[0018] SEQ ID NO.08: 5′-AGTAACGGCAGACTTCTCC-3′;

[0019] SEQ ID NO. 12: 5'-ACTAGCAACCTCAAACAGACACC-3'.

[0020] Optionally, the specific probe 1, specific probe 2, specific probe 3, and specific probe 4 are all connected with fluorescent labeling groups, and the fluorescent labeling groups on the specific probe 1, specific probe 2, specific probe 3, and specific probe 4 generate different fluorescent signals during the PCR process.

[0021] Optionally, the specific probe 1 is connected to a FAM fluorescent labeling group, the specific probe 2 is connected to a HEX fluorescent labeling group, the specific probe 3 is connected to a CY5 fluorescent labeling group, and the specific probe 4 is connected to a ROX fluorescent labeling group.

[0022] In a second aspect of the present application, a kit for human parainfluenza virus typing detection is provided, comprising the composition for human parainfluenza virus typing detection according to any one of claims 1 to 4.

[0023] Optionally, the concentration of each primer of primer pair 1, primer pair 2, primer pair 3, and primer pair 4 in the composition is 0.1-0.4 μM.

[0024] Optionally, the concentration of specific probe 1, specific probe 2, specific probe 3, and specific probe 4 in the composition is 0.05-0.2 μM.

[0025] Optionally, the kit further comprises: a lyophilization protectant, a PCR buffer, a Taq DNA polymerase, a reverse transcriptase, a positive control substance, a negative control substance and a preservation solution.

[0026] Optionally, the preservation solution includes: guanidine thiocyanate, NaCl, KCl, Triton X-100, Nonidet P-40, trehalose, sorbitol, PEG20000, betaine, proteinase K and NaN3.

[0027] Optionally, the buffer comprises: Tris-HCl, MgCl2, dNTPs, (NH4)2SO4, BSA, Tween20, Gelatin, glycerol, DMSO and NaN3.

[0028] Optionally, the lyoprotectant includes: trehalose, sucrose, mannitol and PEG20000.

[0029] The composition and kit for human parainfluenza virus typing detection provided by the present invention, and the primer and probe combination disclosed in the present invention have the advantages of high sensitivity, high specificity, and highly accurate detection results, and the kit of the present invention can meet the detection limit of no more than 200 copies / mL. Furthermore, the present invention innovatively adds a nucleic acid extraction reagent to the preservation solution of the sampling tube, which can eliminate the sample extraction step, and the sample can be directly taken on the machine after sampling, and the rapid amplification can produce results in only 30 minutes. Trehalose, betaine and other PCR enhancer components are preferably used in the preservation solution of the present invention, and the kit made using the composition of the present invention has a strong anti-interference ability. The amplification reagent in the kit provided by the present invention is presented in the form of a lyophilized product, which can be stored at room temperature and has stable performance within 12 months of storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a graph showing the test results of the positive control substance in Example 2 of the present invention;

[0031] Figure 2 This is a graph showing the test results of the negative control substance in Example 2 of the present invention. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The present application provides a composition for human parainfluenza virus typing detection, comprising: a primer pair and a probe corresponding thereto; the primer pair comprises: a primer pair 1 for parainfluenza virus type I, whose nucleotide sequence is shown in SEQ ID NO.01 and SEQ ID NO.02; a primer pair 2 for parainfluenza virus type II, whose nucleotide sequence is shown in SEQ ID NO.03 and SEQ ID NO.04; a primer pair 3 for parainfluenza virus type III, whose nucleotide sequence is shown in SEQ ID NO.05 and SEQ ID NO.06; the probe comprises: a specific probe 1 for parainfluenza virus type I, whose nucleotide sequence is shown in SEQ ID NO.09; a specific probe 2 for parainfluenza virus type II, whose nucleotide sequence is shown in SEQ ID NO.10; and a specific probe 3 for parainfluenza virus type III, whose nucleotide sequence is shown in SEQ ID NO.11.

[0034] Table 1 Sequence information of primers and probes

[0035]

[0036] In the design of primer pairs and corresponding probes, the Tm value of each primer is close to 60°C, the length of the amplified product ranges from 80 to 150 bp, and the Tm value of each probe is close to 70°C. Each pair of primers and probes is tested and optimized for amplification until a smooth amplification curve with a high signal value is obtained. After composite amplification testing, non-specific amplification, primer dimers, and other interactions or cross reactions are not generated.

[0037] Optionally, the primer pair further includes: a primer pair 4 for the human β-globin gene used as an internal reference, whose nucleotide sequence is shown in SEQ ID NO.07 and SEQ ID NO.08; the probe further includes a specific probe 4 for the human β-globin gene used as an internal reference, whose nucleotide sequence is shown in SEQ ID NO.12. This kit uses a specific primer pair and probe designed for the human β-globin gene as an internal standard (IC), and monitors the amplification detection process of the sample by detecting the endogenous β-globin gene to avoid false negative results.

[0038] Table 2 Sequence information of primers and probes

[0039]

[0040] Optionally, the specific probe 1, specific probe 2, specific probe 3, and specific probe 4 are all connected with fluorescent labeling groups, and the fluorescent labeling groups on the specific probe 1, specific probe 2, specific probe 3, and specific probe 4 generate different fluorescent signals during the PCR process.

[0041] Optionally, the specific probe 1 is connected to a FAM fluorescent labeling group, the specific probe 2 is connected to a HEX fluorescent labeling group, the specific probe 3 is connected to a CY5 fluorescent labeling group, and the specific probe 4 is connected to a ROX fluorescent labeling group. The probe uses a highly specific TaqMan probe, which can bind to the corresponding nucleic acid fragment and be hydrolyzed under the action of Taq enzyme exonuclease activity to generate a fluorescent signal. A real-time amplification curve can be obtained based on the relationship between the fluorescent signal and the number of amplification cycles.

[0042] The present application also provides a kit for human parainfluenza virus typing detection, comprising the aforementioned composition for human parainfluenza virus typing detection.

[0043] Optionally, the concentration of each primer of primer pair 1, primer pair 2, primer pair 3, and primer pair 4 in the composition is 0.1-0.4 μM.

[0044] Optionally, the concentrations of specific probe 1, specific probe 2, specific probe 3, and specific probe 4 in the composition are all 0.05-0.2 μM.

[0045] Optionally, the kit further comprises: a lyophilization protectant, a PCR buffer, a DNA polymerase, a reverse transcriptase, a positive control substance, a negative control substance and a preservation solution.

[0046] Optionally, the preservation solution includes: 10-100mM guanidine thiocyanate (preferably 50mM), 10-50mM NaCl (preferably 25mM), 10-50mM KCl (preferably 25mM), 0.1-0.5% (v / v) Triton X-100 (preferably 0.2%), 0.05-5% Nonidet P-40 (preferably 0.2%), 1-10% trehalose (preferably 5%), 0.1-1% sorbitol (preferably 0.2%), 1-10% PEG20000 (preferably 3%), 5-15mg / mL betaine (preferably 10mg / mL), 0.01-0.1mg / mL proteinase K, and 0.01-0.05% NaN3 (preferably 0.01%).

[0047] Optionally, the buffer comprises: 30-70mM Tris-HCl (preferably 50mM), 2-8mM MgCl2 (preferably 3mM), 0.5-5mM dNTPs (preferably 1mM), 5-30mM (NH4)2SO4 (preferably 20mM), 0.01-0.1% (w / v) BSA (preferably 0.05%), 0.01-0.5% (v / v) Tween20 (preferably 0.2%), 0.005-0.05% (w / v) Gelatin (preferably 0.01%), 0.01-0.1% (v / v) glycerol (preferably 0.05%), 0.05-0.5% (v / v) DMSO (preferably 0.2%), and 0.005-0.05% (w / v) NaN3 (preferably 0.01%).

[0048] The DNA polymerase required for the reaction is a hot start DNA polymerase, antibody blocking modified or chemically modified, and each amplification system (25 μL) requires 2.5U to 5U of DNA polymerase. The reverse transcriptase required for the reaction requires 1U to 2U of each amplification system (25 μL).

[0049] The amplification reagent is presented in the form of a lyophilized product and can be stably stored at room temperature for 12 months. The lyophilized protective agent includes: 1-10% (w / v) trehalose (preferably 5%), 5-50% (w / v) sucrose (preferably 10%), 0.5-10% (w / v) mannitol (preferably 2%), and 1-10% (w / v) PEG20000 (preferably 3%).

[0050] The present application is further described in detail below through specific embodiments.

[0051] Example 1 Selection of primer and probe combinations for human parainfluenza virus typing detection kit

[0052] 1. Design principles of primer pairs and probes for human parainfluenza virus typing detection system

[0053] Primer and probe length: Each primer is 16 to 30 bases long, and the fluorescent probe is 20 to 35 bases long; the GC% in primers and probes is required to be 30 to 70%, the primer Tm value is 58 to 62°C, and the probe Tm value is 5 to 10°C higher than the primer Tm value; the 3' ends of the primers and probes themselves and between the primers and probes should avoid base pairing as much as possible to avoid the formation of primer-probe dimers, thereby reducing the working efficiency of the primers and probes; the primers and probes themselves and between the primers and probes should avoid more than 6 pairs of continuous base pairing as much as possible to avoid Avoid the formation of primer-probe dimers, thereby reducing the working efficiency of the primer probe; the length of the target fragment amplified by the upstream and downstream primers is 80 to 200 bp, and the probe is located between the upstream and downstream primers, as close to the upstream primer as possible, and guanine should be avoided at the 5' end of the probe to avoid inhibition of the probe fluorescence, thereby reducing the sensitivity of the detection system; the selected primers and probes are required to be designed in the conserved regions in the genome of Chlamydia pneumoniae, Mycoplasma pneumoniae or respiratory adenovirus, respectively, and can specifically detect the target viral nucleic acid, and have no cross reaction with other species.

[0054] 2. Design of primer pairs and probes for human parainfluenza virus typing detection system

[0055] After homology comparison of the parainfluenza virus type I, type II, type III genome sequences and human β-globin gene sequences retrieved from Genbank, the homologous conserved segments of the HN gene and the internal standard β-globin gene were found, and five sets of primers and probe sets were designed (as shown in Table 3 below) through retrieval analysis using the Blast tool in the GenBank database. The probe of the parainfluenza virus type I HN gene was labeled with a fluorescent reporter group (FAM) at the 5' end and with BHQ2 at the 3' end; the probe of the parainfluenza virus type II HN gene was labeled with a fluorescent reporter group (HEX) at the 5' end and with BHQ2 at the 3' end; the probe of the parainfluenza virus type III HN gene was labeled with a fluorescent reporter group (CY5) at the 5' end and with BHQ2 at the 3' end.

[0056] Table 3 Information of five candidate primer pairs and probes

[0057]

[0058]

[0059] 3. Screening of primer pairs and probes for human parainfluenza virus typing detection system

[0060] The detection limit reference substances of parainfluenza virus type I, type II and type III were selected to screen the sensitivity of five sets of candidate primer pairs and probes. The test results are shown in Table 4 below:

[0061] Table 4 Comparison of detection results of five candidate primer pairs and probes

[0062]

[0063] Comparing the detection results of the five sets of primer pairs and probes, it was found that the first set of primer pairs and probes had higher detection sensitivity and could meet the requirements of high sensitivity.

[0064] Example 2 Preparation of a human parainfluenza virus typing test kit

[0065] 1. Primer pairs and probes for human parainfluenza virus typing detection system

[0066] 1. Primer pairs and probes for human parainfluenza virus typing detection system

[0067] Amplification primer pairs and corresponding probes were designed for parainfluenza virus type I, type II, and type III, respectively. The primer pair probe sequences, primer pair probe concentrations, and fluorescent labeling methods are shown in Table 1. In addition, this kit selects human genome β-globin as an internal standard (IC), and its primer probe information is shown in Table 2.

[0068] 2. Preparation of human parainfluenza virus typing detection kit

[0069] Table 5 Components of the kit

[0070]

[0071]

[0072] Table 6 Components and contents of each 20 μL PCR freeze-dried ball system

[0073] Components Addition volume (μL) PCR reaction system 17.5 Primers and probes 1 Taq DNA polymerase 1 Reverse transcriptase 0.5

[0074] The PCR reaction system includes a buffer and a freeze-drying protective agent.

[0075] Table 7 Concentration of primers and probes in PCR freeze-dried ball system

[0076] Primer / probe Final concentration SEQ ID NO.01 0.1μM SEQ ID NO.02 0.1μM SEQ ID NO.03 0.2μM SEQ ID NO.04 0.2μM SEQ ID NO.05 0.3μM SEQ ID NO.06 0.3μM SEQ ID NO.07 0.4μM SEQ ID NO.08 0.4μM SEQ ID NO.09 0.05μM SEQ ID NO.10 0.1μM SEQ ID NO.11 0.15μM SEQ ID NO.12 0.2μM

[0077] The pseudovirus contained in the positive control is a 1000 bp fragment corresponding to the detection target region, with a concentration of 10 3TU / mL, purchased from Sangon Biotech (Shanghai) Co., Ltd. Hot-start DNA polymerase and reverse transcriptase were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0078] 3. Lyophilization of human parainfluenza virus amplification system

[0079] The reagents were freeze-dried using a freeze dryer (model: LGJ-12S) purchased from Jiangsu Scientific Instruments Co., Ltd. The freeze drying procedure is shown in Table 8 below:

[0080] Table 8 Freeze-drying procedure

[0081]

[0082] Example 3 Application of human parainfluenza virus typing detection kit

[0083] 1. Amplification of positive control

[0084] Dissolve the positive control with 500 μL of sampling solution and mix well for later use, take 65 μL to dissolve the PCR freeze-dried ball prepared in Example 2, mix well and centrifuge briefly to obtain a PCR reaction solution. Place the PCR reaction solution in a fluorescent PCR instrument (the model used in this example is a GN7120 rapid nucleic acid analyzer, obtained from GeneDan Biotechnology Co., Ltd.) for PCR amplification.

[0085] PCR amplification program: pre-denaturation at 97°C for 30 seconds; 97°C for 8 seconds, 57°C for 15 seconds, 42 cycles.

[0086] After amplification, GN7120 was used to analyze the data. The test results are as follows: Figure 1 As shown, the amplification curves of PIVⅠ, PIVⅡ, PIVⅢ and IC are clear, showing a standard S-shaped curve, and there is no obvious fluctuation in the fluorescence background.

[0087] 2. Amplification of negative control

[0088] Dissolve the negative control with 500 μL of sampling solution and mix well for later use. Take 65 μL to dissolve the PCR freeze-dried ball prepared in Example 2, mix well and centrifuge briefly to obtain the PCR reaction solution. Place the PCR reaction solution in a fluorescent PCR instrument (the model used in this example is a GN7120 rapid nucleic acid analyzer, obtained from GeneDan Biotechnology Co., Ltd.) for PCR amplification.

[0089] PCR amplification program: pre-denaturation at 97°C for 30 seconds; 97°C for 8 seconds, 57°C for 15 seconds, 42 cycles.

[0090] After amplification, GN7120 was used to analyze the data. The test results are as follows: Figure 2As shown, the IC amplification curve is clear and presents a standard S-shaped curve. There is no obvious fluctuation in the fluorescence background and no non-specific amplification occurs.

[0091] Example 4 Comparison of human parainfluenza virus typing test kit with commercially available kits

[0092] 1. Sample and commercial kit information

[0093] 20 clinical samples were provided by the Jingzhou Center for Disease Control and Prevention in Hubei Province. Two samples were collected from each sample, one was placed in the preservation solution of the kit of the present invention, and the other was placed in the preservation solution of the comparison kit.

[0094] The comparison kit was a six-item respiratory virus nucleic acid detection kit (fluorescent PCR probe method), national device registration number 20213400567, purchased from Shanghai Berger Medical Technology Co., Ltd.

[0095] 2. Sample Pretreatment

[0096] The cells scraped with an oropharyngeal swab are placed in the preservation solution of each kit. Since a nucleic acid extraction reagent is added to the preservation solution, the kit of the present invention directly takes 65 μL of sample as a PCR amplification template after mixing, without pretreatment. All commercially available kits require a series of pretreatment operations such as centrifugal shaking and lysis, and the pretreatment operation takes about 40 minutes. The kit provided in this application does not require a kit, so it greatly saves the overall detection time and improves detection efficiency.

[0097] 3. Amplification Procedure

[0098] The kit of the present invention adopts a rapid amplification program, and the comparison kit performs amplification according to the program recommended in the instruction manual. The specific amplification program and time are shown in Table 9.

[0099] Table 9 Comparison of amplification procedures with commercial kits

[0100]

[0101] 4. Sample testing

[0102] The PCR reaction solution was prepared according to the sample addition of Example 2 and prepared into a lyophilized product, and then rapid amplification was performed on GN7120, and the GN7120 rapid nucleic acid analyzer was obtained from GeneDan Biotechnology Co., Ltd.; the comparison kit was operated according to its instructions, and the model used was ABI7500. The test results are shown in Table 10. After data statistics, the positive compliance rate of the kit of the present invention was 100%, the negative compliance rate was 100%, and the total compliance rate was 100% compared with the comparison kit.

[0103] Table 10 Comparison of positive and negative test results of the kit

[0104]

[0105]

[0106] Example 5 Performance verification of real-time fluorescence quantitative PCR kit for human parainfluenza virus typing detection

[0107] 1. Precision verification

[0108] The parainfluenza virus enterprise reference material was selected for precision verification, which was produced by GeneDan Biotech Co., Ltd. The concentration of the parainfluenza virus enterprise precision weak positive reference material was 800 copies / mL. Two people (A and B) tested it for 20 days, repeated twice a day, and obtained the coefficient of variation CV. The model used for this precision verification was the GN7120 rapid nucleic acid analyzer, which was obtained from GeneDan Biotech Co., Ltd.

[0109] The test results are shown in Table 11. According to data statistics, the CV of the weak positive reference tested by the kit of the present invention is less than 5%.

[0110] Table 11 Precision verification of real-time fluorescence quantitative PCR kit for human parainfluenza virus typing detection

[0111]

[0112]

[0113] Based on the above experiments, 20 days of testing were carried out under the same conditions, with 2 analysis batches per day, and each batch was repeated twice by different people. A total of 80 test results showed that the detection precision of the real-time fluorescence quantitative PCR kit for human parainfluenza virus typing detection was within 5%.

[0114] 2. Detection limit verification

[0115] The parainfluenza virus enterprise reference product was selected for detection limit verification, which was produced by GeneDan Biotech Co., Ltd. The parainfluenza virus enterprise detection limit reference product was gradient diluted to 200 copies / mL and repeated 20 times. The model used for this detection limit verification was the GN7120 rapid nucleic acid analyzer, which was obtained from GeneDan Biotech Co., Ltd.

[0116] The test results are shown in Table 12. According to data statistics, the kit of the present invention can meet the detection limit of no more than 200 copies / mL.

[0117] Table 12 Verification of detection limit of real-time fluorescence quantitative PCR kit for human parainfluenza virus typing detection

[0118]

[0119]

[0120] 3. Validation of analytical specificity

[0121] The samples tested are bocavirus, Mycoplasma pneumoniae, adenovirus (types 3 and 7), human parainfluenza virus (type 4), influenza A / B virus, respiratory syncytial virus, herpes simplex virus type 1, cytomegalovirus, human coronavirus, enterovirus 71, human metapneumovirus, measles virus, Neisseria, varicella-zoster virus, mumps virus, rhinovirus, Epstein-Barr virus, Staphylococcus aureus, Bordetella pertussis, Staphylococcus epidermidis, non-toxic Mycobacterium tuberculosis, Neisseria meningitidis, Chlamydia pneumoniae, Streptococcus pneumoniae, Pneumocystis jiroveci, Moraxella catarrhalis, Streptococcus pyogenes, Corynebacterium, Streptococcus salivarius, Candida albicans, Lactobacillus, Haemophilus influenzae, Legionella pneumophila, and Klebsiella pneumoniae.

[0122] Using healthy people's throat swab samples, cross-reacting pseudoviruses and bacteria were diluted to 10 5 PFU / ml and 10 6 CFU / ml.

[0123] Pseudoviruses and bacteria used in cross-reactions were purchased from Nanjing Standa Biotechnology Co., Ltd.

[0124] The model used for this analysis specificity verification was the GN7120 rapid nucleic acid analyzer, which was obtained from GeneDx Biotechnology Co., Ltd.

[0125] Cross-reactivity evaluation is divided into two aspects, including gene sequence alignment and cross-reactivity verification.

[0126] a) Gene sequence alignment

[0127] The whole genome sequence of the cross-reactive sample was obtained from NCBI, and clustered and compared with the target sequences of HN gene of parainfluenza virus type I, type II, and type III. The test results are shown in Table 13. After data statistics, the target sequences of HN gene of parainfluenza virus type I, type II, and type III in the kit of the present invention have no homology with the gene sequences of bacteria and pathogens of the respiratory tract.

[0128] Table 13 Gene sequence comparison results

[0129]

[0130]

[0131] b) Verification of cross-reactivity

[0132] The kit of the present invention was used for detection, and the detection results are shown in Table 14. According to data statistics, common respiratory bacteria and pathogens have no cross-interference with the kit of the present invention.

[0133] Table 14 Cross-reaction verification results

[0134]

[0135]

[0136] 4. Interference studies

[0137] The test substances selected for the interfering substance study are shown in Table 15:

[0138] Table 15 Interfering substances

[0139]

[0140] Using throat swab samples from healthy people containing different interfering substances, the parainfluenza virus detection limit reference substance was diluted to the detection limit concentration (200 copies / mL) and the measurement was repeated 20 times.

[0141] The parainfluenza virus corporate reference product was produced by GeneDx Biotechnology Co., Ltd.

[0142] The model used in this interference study was the GN7120 rapid nucleic acid analyzer, which was obtained from GeneDx Biotechnology Co., Ltd.

[0143] Table 16 Interference substance test results

[0144]

[0145]

[0146] The present invention uses the typing test of parainfluenza virus, and can detect parainfluenza virus type I, type II, and type III in one sampling, so that screening and diversion can be completed in one step; the nucleic acid extraction reagent is innovatively added to the sampling tube, which can avoid the sample extraction step, and the sample can be directly taken on the machine after sampling, and the rapid amplification can produce the result in only 30 minutes. Compared with other test kits on the market, the time is greatly shortened. For specific data, see Table 17, and the sensitivity is high, and the detection result is highly accurate.

[0147] Table 17 Comparison of detection time with mainstream commercial nucleic acid detection kits on the market

[0148]

[0149]

[0150] 5. Stability verification

[0151] The amplification reagent in the kit provided by the present invention is presented in the form of a lyophilized product, can be stored at room temperature, and is stable within 12 months.

[0152] The test kit was stored at 28°C and the test kit within one month of its expiration date was used for verification of detection limit and repeatability.

[0153] Inspection standard:

[0154] 1) Each target detection limit sample was tested 20 times, and the results were all positive;

[0155] 2) Repeatability test: CV of each target was ≤5%.

[0156] The test results are shown in Tables 18 and 19 below.

[0157] Table 18 Detection limit test results of the kit within 1 month of expiration

[0158]

[0159]

[0160] Table 19 Repeatability test results of the kit within 1 month of expiration date

[0161]

[0162] The present invention provides a composition for parainfluenza virus typing detection and a real-time fluorescence quantitative PCR kit, which are applied to the detection of parainfluenza virus type I, type II, and type III, and have the characteristics of strong fluorescence signal and high sensitivity. The present invention adopts a probe group composed of three probes, rather than three parallel technical solutions, so the gene that can achieve simultaneous amplification detection is selected, and it is necessary to consider the factors that cause the failure of detection due to the reaction between the template sequence and the probe, and the influence of the probe sequence on specificity, sensitivity and accuracy. It is necessary to select a specific design idea to overcome the difficulties and key points in the actual operation of the multiplex PCR detection technology, so as to obtain the technical solution of the present application.

[0163] The kit of the present invention has a wide coverage for detecting respiratory pathogens. After sample collection, no extraction is required and the test can be directly performed on the machine. The PCR reaction can be completed within 30 minutes, and the analysis specificity is high, and the sensitivity can reach 200 copies / mL. The amplification reagent in the kit provided by the present invention is presented in the form of a lyophilized product and can be stably stored at room temperature for 12 months.

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for human parainfluenza virus typing detection, characterized in that: include: Primer pairs and corresponding probes; The primer pairs include: primer pair 1 for parainfluenza virus type I, whose nucleotide sequences are shown in SEQ ID NO.01 and SEQ ID NO.02; primer pair 2 for parainfluenza virus type II, whose nucleotide sequences are shown in SEQ ID NO.03 and SEQ ID NO.04; primer pair 3 for parainfluenza virus type III, whose nucleotide sequences are shown in SEQ ID NO.05 and SEQ ID NO.06; The probes include: a specific probe 1 for parainfluenza virus type I, whose nucleotide sequence is shown in SEQ ID NO.09; a specific probe 2 for parainfluenza virus type II, whose nucleotide sequence is shown in SEQ ID NO.10; a specific probe 3 for parainfluenza virus type III, whose nucleotide sequence is shown in SEQ ID NO.11; SEQ ID NO.01: 5′-TAGARGCAGCATACACTACTACATCRT-3′; SEQ ID NO.02: 5′-GGCTGGTTTGGTTGATTTCA-3′; SEQ ID NO.03: 5′-TCCTATGTCATCCGTTATCACTCA-3′; SEQ ID NO.04: 5′-CCATCATGACTTGGGAATTGTTAA-3′; SEQ ID NO.05: 5′-GTGTCATCTGTCATATTAGACTCACAA-3′; SEQ ID NO.06: 5′-GCTGAGAGTGTTCTATTTCGGAT-3′; SEQ ID NO.09: 5′-TCACTCAYTTCGGAAAGGGCTACTGC-3′; SEQ ID NO.10: 5′-GAGTGCTGTCTYATTTGTCCATTGTCTG-3′; SEQ ID NO. 11: 5'-AACCCAGTCATAACTTACTCAACAGCAACC-3'.

2. A composition for human parainfluenza virus typing detection according to claim 1, characterized in that: The primer pair also includes: a primer pair 4 for the human β-globin gene used as an internal reference, whose nucleotide sequence is shown in SEQ ID NO.07 and SEQ ID NO.08; the probe also includes a specific probe 4 for the human β-globin gene used as an internal reference, whose nucleotide sequence is shown in SEQ ID NO.12; SEQ ID NO.07: 5′-GCTTACATTTGCTTCTGACA-3′; SEQ ID NO.08: 5′-AGTAACGGCAGACTTCTCC-3′; SEQ ID NO. 12: 5'-ACTAGCAACCTCAAACAGACACC-3'.

3. A composition for human parainfluenza virus typing detection according to claim 2, characterized in that: The specific probe 1, specific probe 2, specific probe 3, and specific probe 4 are all connected with fluorescent labeling groups, and the fluorescent labeling groups on the specific probe 1, specific probe 2, specific probe 3, and specific probe 4 generate different fluorescent signals during the PCR process.

4. A composition for human parainfluenza virus typing detection according to claim 3, characterized in that: The specific probe 1 is connected to a FAM fluorescent labeling group, the specific probe 2 is connected to a HEX fluorescent labeling group, the specific probe 3 is connected to a CY5 fluorescent labeling group, and the specific probe 4 is connected to a ROX fluorescent labeling group.

5. A kit for human parainfluenza virus typing detection, characterized in that: A composition for human parainfluenza virus typing detection comprising any one of claims 1 to 4.

6. A kit for human parainfluenza virus typing detection according to claim 5, characterized in that: The concentration of each primer of primer pair 1, primer pair 2, primer pair 3 and primer pair 4 in the composition is 0.1-0.4 μM.

7. A kit for human parainfluenza virus typing detection according to claim 5, characterized in that: The concentrations of specific probe 1, specific probe 2, specific probe 3 and specific probe 4 in the composition are 0.05-0.2 μM.

8. A kit for human parainfluenza virus typing detection according to claim 5, characterized in that: The kit also includes: a freeze-dried protective agent, a PCR buffer, a Taq DNA polymerase, a reverse transcriptase, a positive control substance, a negative control substance and a preservation solution.

9. A kit for human parainfluenza virus typing detection according to claim 8, characterized in that: The preservation solution includes: guanidine thiocyanate, NaCl, KCl, Triton X-100, Nonidet P-40, trehalose, sorbitol, PEG20000, betaine, proteinase K and NaN3.

10. A kit for human parainfluenza virus typing detection according to claim 8, characterized in that: The buffer comprises: Tris-HCl, MgCl2, dNTPs, (NH4)2SO4, BSA, Tween20, Gelatin, glycerol, DMSO and NaN3.

11. A kit for human parainfluenza virus typing detection according to claim 8, characterized in that: The freeze-drying protective agent includes: trehalose, sucrose, mannitol and PEG20000.

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