CN113512611B - Gene film chip-based method for detecting various respiratory viruses - Google Patents
Gene film chip-based method for detecting various respiratory viruses Download PDFInfo
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Abstract
The invention discloses a method for detecting various respiratory viruses based on a gene membrane chip, which comprises the following detection steps of: taking a nose/throat secretion swab into a preservation tube containing sample preservation liquid, fully washing, sucking all the liquid into a new centrifuge tube, centrifuging, and discarding the supernatant; nucleic acid extraction: adding the extracting solution and proteinase K into the centrifuge tube, heating and centrifuging after vortex oscillation, and taking supernatant for PCR reaction; multiplex PCR (polymerase chain reaction) sample addition and amplification: taking a multiplex respiratory virus PCR Mix, a nucleic acid extracting solution and enzyme-free water to carry out multiplex PCR sample adding and amplification; membrane chip hybridization: carrying out hybridization reaction on multiple PCR amplification products and a membrane chip by a full-automatic membrane chip nucleic acid molecule hybridization instrument; and (3) sending a detection result report: and directly reporting the detection result by a full-automatic membrane chip nucleic acid molecule hybridization instrument automatic analysis system. The invention combines multiple PCR and reverse dot hybridization technologies, can realize single reaction, detect multiple respiratory pathogens in a single sample at the same time and automatically send out a detection result report.
Description
Technical Field
The invention relates to a method for detecting various respiratory viruses based on a gene membrane chip, and belongs to the technical field of virus detection.
Background
Respiratory tract infection is one of the common clinical diseases, and main pathogens include bacteria, viruses, mycoplasma, chlamydia, fungi and the like, wherein more than 90% of the respiratory tract infection is caused by respiratory tract virus infection, acute respiratory tract infection is one of the most important threats of children, the respiratory tract infection is frequently caused by children, the elderly and immunocompromised persons, influenza A virus, influenza B virus, respiratory syncytial virus, respiratory adenovirus, rhinovirus, parainfluenza virus and the like are common, most of the respiratory tract infection has strong infectivity, and the respiratory tract infection has fast transmission, short latency period, acute morbidity and similar clinical manifestations, and patients all show fever and upper respiratory tract infection symptoms, so that the infectious pathogens are difficult to distinguish from the clinical manifestations alone.
The detection method commonly used at present comprises the following steps: virus routine culture, serological test, antigen-antibody detection and molecular biology method (fluorescent polymerase chain reaction method and gene chip method); most viruses are detected by virus culture and serological detection, the former is generally regarded as a "gold standard" for diagnosis, but the requirement of cell separation on culture technology is high, and the time is long, so that early diagnosis is not favored.
Although the serological detection method is convenient and easy to implement, the detection of double serum needs a longer time, the detection result cannot be applied to clinic in time, and the serological detection result is easily influenced by the immunity level of the organism.
The antibody antigen detection method comprises an immunofluorescence method, an enzyme-linked immunosorbent assay method and the like, and the detection speed of the method is high, but the sensitivity of the method is low, and the time interval between virus transmission and antibody appearance is long, so that the method is not beneficial to early diagnosis of infection caused by micro-infection.
With the development of molecular biology technology, respiratory pathogen nucleic acid detection technology has been developed vigorously, and at present, a fluorescent quantitative polymerase chain reaction technology is mainly used for screening and confirming patients infected by suspected respiratory pathogens clinically, but the method is long in manual operation time, and is difficult to detect multiple pathogens in a single reaction, and a part of products or methods which can detect multiple pathogens simultaneously are not only in the following two realization forms: firstly, the number of fluorescent channels is limited, the number of the fluorescent channels is not more than 4, if the number of the fluorescent channels is more than 4, the mutual interference of the fluorescent channels can occur, false negative or false positive results can occur, if more than 4 indexes are needed, the detection is performed by a system which is divided into 2 or more, compared with the detection of the single system, the price of the reagent is increased by times, the workload is also greatly increased, and secondly, the detection of the single system is performed by detecting one index in 96 or 384 pore plates, 30-100 targets (including quality control and 3 repetition of a single sample) can be detected simultaneously, but the method has the same problems, the cost of the reagent is increased by times, and the workload is extremely high.
Besides the fluorescent quantitative polymerase chain reaction method, the gene chip method is also a currently used molecular biology detection method, the problem of low detection flux of the fluorescent quantitative polymerase chain reaction technology is perfectly solved, and the flux can reach hundreds or even thousands of targets, but due to high price of equipment, consumables and the like, the method is more used in scientific research at present, and the marketization degree is lower.
Disclosure of Invention
The invention mainly aims to provide a method for detecting various respiratory viruses based on a gene membrane chip, which solves the problems of low respiratory virus detection speed, long time consumption, low sensitivity, high cost and difficulty in detecting various pathogens in a single reaction.
The aim of the invention can be achieved by adopting the following technical scheme:
a method for detecting various respiratory viruses based on a gene membrane chip comprises the following detection steps:
(1) Sample pretreatment;
(2) Extracting nucleic acid;
(3) Multiple PCR sample addition and amplification;
(4) Membrane chip hybridization;
(5) And (5) sending out a detection result report.
Preferably, the detection method comprises simultaneous detection of 6 common respiratory viruses by multiplex PCR combined with reverse dot hybridization, and monitoring nucleic acid extraction with GAPDH (glyceraldehyde-3-phosphate dehydrogenase gene) as internal control, PCR process, PC (Positive control) and NC (Negative control) as chip quality control.
Preferably, the common respiratory viruses include influenza a virus (hereinafter referred to as a flow), influenza b virus (hereinafter referred to as b flow), respiratory syncytial virus (hereinafter referred to as syncytial virus), adenovirus, rhinovirus, and parainfluenza virus;
Wherein, the forward primer sequence of the first stream is shown as SEQ ID No.4, and the reverse primer sequence is shown as SEQ ID No. 5; the probe sequence is shown as SEQ ID No. 6;
The forward primer sequence of the second flow is shown as SEQ ID No.7, and the reverse primer sequence is shown as SEQ ID No. 8; the probe sequence is shown as SEQ ID No. 9;
the forward primer sequence of the syncytial virus is shown as SEQ ID No.10, and the reverse primer sequence is shown as SEQ ID No. 11; the probe sequence is shown as SEQ ID No. 12;
the forward primer sequence of adenovirus is shown as SEQ ID No.13, and the reverse primer sequence is shown as SEQ ID No. 14; the probe sequence is shown as SEQ ID No. 15;
The forward primer sequence of the rhinovirus is shown as SEQ ID No.16, and the reverse primer sequence is shown as SEQ ID No. 17; the probe sequence is shown as SEQ ID No. 18;
The forward primer sequence of the parainfluenza virus is shown as SEQ ID No.19, and the reverse primer sequence is shown as SEQ ID No. 20; the probe sequence is shown as SEQ ID No. 21.
Preferably, the sample processing specifically includes: nasal/pharyngeal secretion swabs were taken in sample holding tubes, after vortexing, all solutions were transferred to new centrifuge tubes and centrifuged for 5min, and the supernatant was discarded.
Preferably, the nucleic acid extraction specifically comprises: adding nucleic acid extract and proteinase K into a centrifuge tube, vortex shaking for 15s, heating in a metal bath with constant temperature of 56 ℃ for 10-15min, adjusting the temperature to 95 ℃ and heating for 2min, centrifuging for 5min, and taking supernatant to be used as nucleic acid solution participating in PCR reaction.
Preferably, the multiplex PCR sample addition and amplification are specifically: adding components of a PCR amplification system into a centrifuge tube, fully mixing to obtain a multiplex respiratory virus PCR Mix, then adding the multiplex respiratory virus PCR Mix, a nucleic acid solution and double distilled water into a PCR tube to prepare a PCR amplification solution, and finally amplifying the PCR amplification solution to obtain a PCR amplification product.
Preferably, the membrane chip hybridization specifically comprises: step ①, adding the PCR amplification product into a membrane chip containing hybridization solution, and heating at 45 ℃ for 30min; step ②, adding hybridization cleaning solution, and heating at 52 ℃ for 3min; step ③, repeating step ② once; step ④, adding an enzyme-labeled solution, and heating at 42 ℃ for 10min; step ⑤, adding an enzyme-labeled cleaning solution I, and heating at 42 ℃ for 3min; step ⑥, adding an enzyme-labeled cleaning solution II, and heating at 37 ℃ for 3min; step ⑦, repeating step ⑥ once; step ⑧, adding a color development liquid, and heating at 37 ℃ for 10min; step ⑨, adding a chromogenic cleaning solution, heating at 37 ℃ for 1min, step ⑩, and repeating step ⑨ twice, wherein the reaction solution is removed by centrifugation after each heating step in step ①、②、④、⑤、⑥、⑧、⑨.
Preferably, the components of the PCR amplification system are UNG enzyme, dATP, dTTP, dCTP, dGTP, dUTP, taq DNA polymerase, reverse transcriptase, PCR buffer solution and primers, wherein the PCR buffer solution is mainly magnesium ion solution, and also comprises potassium ion and betaine; the multiplex PCR system comprises a section of Pos-oligo sequence which is complementary with the PC probe and contains 5' -biotin modification, and is used for hybridization with the PC probe and quality control hybridization process.
Preferably, the influenza a virus comprises subtype a H1N1-2009, seasonal influenza a H1N1, influenza a H3N2, influenza a H7N9 subtype, the influenza b virus comprises subtype b Yamagata, victoria, the respiratory syncytial virus comprises subtype respiratory syncytial virus A, B, the adenovirus comprises human adenovirus subtype 3, 4, 7, 55, and the parainfluenza virus comprises human parainfluenza virus subtype 1,2, 3.
Preferably, the hybridization solution has a composition of 2X SSPE,0.1%SDS,30% deionized formamide.
The hybridization washes consisted of 2 XSSPE, 0.5% SDS.
The enzyme-labeled solution has the composition of 2X SSPE,0.5%SDS,0.4 mug/mL AP-SA.
The enzyme-labeled washing liquid I consists of 2 XSSPE and 0.5% SDS.
The enzyme-labeled cleaning solution II comprises 1M Tris-HCl,5M NaCl,1M MgCl 2.
The color development liquid comprises 0.15mg/mL BCIP,0.30mg/mL NBT,100mmol/L Tris-HCl and 5mmol/L MgCl 2.
The composition of the chromogenic cleaning solution is 1 XPBS.
The beneficial technical effects of the invention are as follows:
the method for detecting the various respiratory viruses based on the gene membrane chip has the following beneficial effects:
1. The method for detecting the multiple respiratory viruses based on the gene film chip realizes screening of detecting the multiple respiratory viruses in a single respiratory sample by combining multiple PCR and reverse spot hybridization technologies, can realize single reaction and simultaneously detect multiple respiratory pathogens in the sample by combining a full-automatic nucleic acid molecule hybridization instrument and a hybridization reagent system matched with the full-automatic nucleic acid molecule hybridization instrument, is simple to operate and high in detection sensitivity, can complete detection of 1-24 samples within 4 hours, and greatly saves detection reagent cost and labor cost.
2. The gene film chip based multiple respiratory tract virus detecting method has the advantages that the sequence containing uracil in the PCR product can be effectively degraded by using UNG enzyme, dUTP and dTTP are mixed according to a certain proportion when the PCR reaction liquid is prepared, so that the amplified product contains deoxyuracil, and the product is sensitive to the UNG enzyme, so that a newly prepared reaction system can be treated by the UNG enzyme before PCR, and aerosol pollution can be prevented to a certain extent.
3. According to the gene membrane chip-based method for detecting various respiratory viruses, the specificity and accuracy of detection results are ensured by taking GAPDH as an internal control to monitor the nucleic acid extraction and PCR process and taking PC and NC as chip quality control to monitor the hybridization process.
Drawings
Fig. 1 is an overall lattice diagram of the present invention.
FIG. 2 is a diagram showing the results of the primer ratio debugging test of the present invention.
FIG. 3 is a system performance test lattice diagram.
FIG. 4 is a graph showing the result of the specific detection of the present invention
FIG. 5 is a graph showing the detection limit of the present invention.
FIG. 6 is a graph showing the results of stability test according to the present invention.
FIG. 7 shows a test result report template obtained by the test method of the present invention.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Embodiment Gene film chip-based method for detecting various respiratory viruses
S1, sequence analysis and primer design: selecting a sequence with specificity of a detection species and large difference with other species as a target fragment according to a target detection target, designing an alternative Primer, wherein the selected method is to search related data and specific sequences, and performing sequence analysis by using related software or databases such as Primer, oligo, NCBI Blast and the like, which is different from a common single Primer system, so long as the single Primer has high amplification efficiency, attention is paid when designing a multiple reaction Primer, and the conditions of interaction among different primers, primer mismatch, primer competition and the like are considered except the condition of the Primer itself;
S2, primer effect test: the method comprises the steps of testing the amplification efficiency of a primer by using a standard template, selecting the primer with high amplification efficiency, and then carrying out a specific test on the selected primer, wherein the specific test is realized by selecting the primer with high amplification efficiency and no non-specific amplification of an amplification non-target template, and the specific test is realized by the following steps: screening primers by taking a non-target template, such as a detection target as a first stream, amplifying the non-target templates of B stream, syncytial virus, adenovirus, rhinovirus, parainfluenza virus, mycoplasma pneumoniae, rickettsia, chlamydia pneumoniae and the like by using the first stream primer, and performing electrophoresis detection to select the primers with high amplification efficiency and no non-specific amplification; the final primer sequences are shown in Table 1:
Table 17 common respiratory tract virus primer sequences
The PC and Pos-oligo sequences are shown in Table 2:
TABLE 2 quality control probe sequences
The PCR amplification system comprises the following components: UNG enzyme, dATP, dTTP, dCTP, dGTP, dUTP, taq DNA polymerase, reverse transcriptase, PCR buffer (Mg 2+), primer, nucleic acid template and double distilled water, wherein the PCR buffer is mainly magnesium ion solution, and also comprises potassium ion, betaine and the like; the specific formulation of each component is shown in table 3 below:
TABLE 3PCR amplification System Components and proportions (50. Mu.L System)
The PCR amplification system components and the ratio (50. Mu.L system) amplification procedure are shown in Table 4:
TABLE 4PCR amplification procedure
Reverse dot hybridization technique;
S3, sequence analysis and hybridization probe design: carrying out product sequence analysis by utilizing related software or databases such as Primer, oligo, DNAman, DNAStar and the like according to target detection targets, particularly amplified product sequences, and designing specific hybridization probes according to hybridization probe design related principles;
Testing hybridization effect;
S4, membrane chip processing: cutting a negatively charged nylon membrane into a size of 20 multiplied by 24cm, and printing a square form with a side length of 11.5cm on the membrane by using a printer; to 94mL of 1 XPBS solution was added 6mL of 50% glutaraldehyde solution; soaking the printed film in PBS solution containing glutaraldehyde, slowly shaking and incubating for 2 hours by a shaking table, and pouring out the solution; washing the membrane with 1 XPBS for 4 times, and incubating for 5 minutes with each shaking; finally, the film is placed in air for drying and then sealed or stored in a vacuum dryer;
S5, probe spotting: the concentration of the probe was determined according to Nanodrop, and the probe was diluted to 15. Mu.M with NaHCO 3 solution having been added with an appropriate amount of ponceau dye and pH 8.4 to a total volume of 70/100. Mu.L (volume 70. Mu.L when the spotting amount was 155. Mu.L, volume 100. Mu.L when the spotting amount was 155X 2); respectively adding probes into appointed 384-hole spot plates on a spot sample instrument according to the spot sample sequence, calling the prepared spot sample program according to the number of the rows and the columns of the spot sample chip, and adjusting the initial position and other parameters of the spot sample needle to sample; after the sample application is finished, the chip is placed in an oven at 80 ℃ to be baked for 1.5 hours so as to enhance the crosslinking of the probe and the membrane;
s6, deactivating: the nylon membrane with the probes is soaked in 0.1M NaOH solution, taken out after 10 minutes, placed in hybridization solution (2 XSSPE, 0.1% SDS) preheated at 60 ℃ for 5 minutes, and finally placed in an oven at 60 ℃ for drying for 1.5 hours;
S7, hybridization of a membrane chip: respectively amplifying the screened primers (corresponding primers are subjected to biotin labeling) and then hybridizing, and automatically analyzing the hybridization effect according to a membrane chip nucleic acid molecular hybridization instrument, wherein a probe with a hybridization probe gray value of more than or equal to 60 and no color development of a non-target probe is selected as an alternative probe;
the hybridization reagent and the flow are as follows:
Step 1, thermally denaturing a PCR product at 95 ℃ for 5min, placing the PCR product on ice, cooling, transferring the PCR product into a hybridization tube containing hybridization liquid, uniformly mixing, transferring all the solutions in the hybridization tube into a membrane chip, and heating at 45 ℃ for 30min; the hybridization solution comprises the following components: 2 XSSPE, 0.1% SDS and 30% deionized formamide;
Step 2, adding hybridization cleaning liquid into the membrane chip, and heating for 3min at 52 ℃; the hybridization cleaning solution comprises the following components: 2 XSSPE and 0.5% SDS;
Step 3, repeating the step 2 once;
step 4, adding an enzyme-labeled solution into the membrane chip, and heating at 42 ℃ for 10min; the enzyme-labeled liquid comprises the following components: 2 XSSPE, 0.5% SDS, and 0.4. Mu.g/mL AP-SA, ready for use;
step5, adding an enzyme-labeled cleaning solution I into the membrane chip, and heating at 42 ℃ for 3min; the enzyme-labeled cleaning solution I comprises the following components: 2 XSSPE and 0.5% SDS;
step6, adding an enzyme-labeled cleaning solution II into the membrane chip, and heating for 3min at 37 ℃; the enzyme-labeled cleaning solution II comprises the following components: 1M Tris-HCl, 5M NaCl and 1M MgCl 2;
step 7, repeating the step 6 once;
Step 8, adding a color developing solution into the film chip, and heating at 37 ℃ for 10min; the color development liquid comprises the following components: 0.15mg/mL BCIP, 0.30mg/mL NBT, 100mmol/L Tris-HCl, 5mmol/L MgCl 2;
Step 9, adding a developing cleaning solution into the film chip, and heating at 37 ℃ for 1min; the color development cleaning liquid comprises the following components: 1 x PBS;
Step 10, repeating the step 9 twice;
Wherein, the reaction liquid is removed by centrifugation after each heating reaction of the steps 1,2, 4, 5, 6, 8 and 9.
The volumes of the hybridization solution, the hybridization cleaning solution, the enzyme-labeled cleaning solution I, the enzyme-labeled cleaning solution II, the color development solution and the color development cleaning solution are all 150-250 mu L, preferably 200 mu L.
The reagents and schemes are shown in table 5:
TABLE 5 hybridization detection procedure
| Sequence number | Hybridization reagent | Hybridization procedure | Reaction temperature | Reaction time | Cycle number |
| 1 | Buffer A (hybridization solution) | Hybridization | 45℃ | 30min | 1 |
| 2 | Buffer B (hybridization cleaning solution) | Hybridization washes | 52℃ | 3min | 2 |
| 3 | Buffer C (enzyme-labeled liquid) | Enzyme label | 42℃ | 10min | 1 |
| 4 | Buffer D (enzyme-labeled cleaning solution I) | ELISA cleaning 1 | 42℃ | 3min | 1 |
| 5 | Buffer E (enzyme-labeled cleaning solution II) | ELISA washing 2 | 37℃ | 3min | 2 |
| 6 | Buffer F (color developing solution) | Color development | 37℃ | 10min | 1 |
| 7 | Buffer G (chromogenic cleaning liquid) | Color development cleaning | 37℃ | 1min | 3 |
The final selected probe sequences are shown in table 6:
TABLE 6 viral probe sequences
S8, system optimization: preparing multiplex PCR primer Mix by using each primer screened by the screening method with 0.2 mu M as initial working concentration, then using each target template to test the system effect according to the detection flow and debug the proportion of the primers, and then according to the whole detection result (as shown in figure 2, (1) is 0.2 mu M/0.2 mu M of the first stream, (2) is 0.6 mu M/0.4 mu M of the first stream, (3) is 0.2 mu M/0.2 mu M of the adenovirus, (4) is 0.2 mu M/0.3 mu M of the adenovirus, and (5) is 0.2 mu M/0.2 mu M of the rhinovirus, and (6) is 0.2 mu M/0.3 mu M of the rhinovirus, respectively debugging the concentration of the target primers; the final primer concentrations after adjustment are shown in Table 7:
TABLE 7 concentration of viral primers
S9, sample pretreatment: taking a nasal/pharyngeal secretion swab in a 1mL sample preservation tube containing sample preservation liquid, sufficiently washing by vortex oscillation for 15s, transferring all the liquid into a 1.5mL centrifuge tube, centrifuging at 12000rpm for 5min, and discarding the supernatant;
s10, extracting nucleic acid: 50. Mu.L of the nucleic acid extract, 3. Mu.L of proteinase K, vortexing for 15s, heating in a metal bath at 56℃for 10-15min, and then heating at 95℃for 2min were added to the centrifuge tube. Centrifuging at 12000rpm for 5min, taking the supernatant as a nucleic acid solution, and performing multiplex PCR amplification reaction;
S11, PCR amplification: preparing a multiplex respiratory tract virus PCR Mix according to the components and the proportion of a PCR amplification system, performing multiplex PCR sample adding and amplification, wherein the PCR amplification system is a 50 mu L system, and the primer Mix is prepared according to the adjusted final concentration of each primer;
the multiple PCR sample adding process comprises the following steps:
1. firstly, adding 26 mu L of multiple respiratory tract virus PCR Mix into a PCR tube;
2. adding 2-5 mu L of the nucleic acid solution obtained in the step S10 into the PCR tube;
3. finally, the PCR tube was filled with double distilled water to a total volume of 50. Mu.L.
Multiplex PCR addition (50. Mu.L) the volumes of the components are shown in Table 8:
TABLE 8 multiplex PCR sample addition volumes of the components
| Sequence number | Component name | Add volume/system | Remarks |
| 1 | Multiplex respiratory virus PCR Mix | 26μL | |
| 2 | Sample nucleic acid | 2-5μL* | |
| 3 | ddH2O | Make up 50 mu L |
The sample nucleic acid is a sample primary extract, the added volume is generally 5% -10% of the reaction system, and excessive PCR inhibitor can be introduced when the added volume is too large, so that the PCR reaction is affected. The corresponding parameter settings in the multiplex PCR amplification procedure are shown in table 9.
TABLE 9 multiplex PCR amplification procedure
S12, hybridization of a membrane chip: performing membrane chip hybridization detection by using the PCR amplification product;
S13, judging a detection result: GAPDH is used as reference control to monitor nucleic acid extraction and PCR process, PC and NC are used as chip quality control to monitor hybridization process, and the result is shown in figure 2 by combining automatic interpretation of instrument and outputting detection result.
In this embodiment, the target detection targets are a-stream, b-stream, syncytial virus, adenovirus, rhinovirus, parainfluenza virus. The first stream includes a first stream H1N1-2009, a seasonal first stream H1N1, a first stream H3N2, a first stream H7N9 subtype, the second stream includes a second stream Yamagata, victoria subtype, the respiratory syncytial virus includes a respiratory syncytial virus A, B subtype, the adenovirus includes a human adenovirus 3, 4, 7, 55 subtype, and the parainfluenza virus includes a human parainfluenza virus 1,2,3 subtype.
Furthermore, the specific method for testing the amplification efficiency of the primer by using the standard template in S2 is to select templates with 1-2 concentration gradients above the critical template concentration for testing, for example, the detection limit of the research and development purpose of the kit is 1.0E+03copies/mL, then the primer is tested by selecting templates with the concentration of 1.0E+04-1.0E+05copies/mL, the detection method is gel electrophoresis, and the concentration of the target product is preferably above 50 ng/mu L.
Furthermore, the specific method for monitoring the hybridization process by using PC and NC as chip quality control is as follows: designing a section Positive Control Probe (positive control probe) irrelevant to target sequences, spotting the section Positive Control Probe (positive control probe) and each detection target probe on a membrane chip, and adding a section of Pos-oligo oligonucleotide which is complementary matched with the positive control probe and is provided with biotin modification into a system to serve as positive quality control in the hybridization reaction process; in addition, a section Negative Control Probe (negative control probe) which is irrelevant to the target sequence is also designed, and the section Negative Control Probe, the detection target probes and the positive control probes are spotted on a membrane chip together to serve as negative quality control in the hybridization reaction process.
Notably, each target primer in S8 comprises a primer modified with 5' biotin.
Experimental example: according to the established detection system, performing specificity test, detection limit and stability test, and testing the detection performance of the system, wherein the probe layout is shown in figure 3;
specificity test: amplifying nucleic acids of a first stream, a second stream, a syncytial virus, an adenovirus, a rhinovirus, a parainfluenza virus, a mycoplasma pneumoniae, rickettsia, and a chlamydia pneumoniae respectively using the adjusted multiplex PCR primer Mix; the hybridization test result is shown in fig. 4, and the percentage of the samples with negative detection in healthy people to the total number of healthy people can be rapidly analyzed;
Limit of detection: carrying out multiple PCR sample adding, amplification and hybridization detection by using IA-1.0E+03copies/mL、IB-1.0E+04copies/mL、RSV-1.0E+04copies/mL、HAdV-1.0E+04copies/mL、HRV-1.0E+04copies/mL、HPIV-1.0E+04copies/mL critical concentration viral nucleic acid respectively; the results are shown in FIG. 5;
Stability test: stability tests were performed on samples of limited concentrations for a-1.0E+03copies/mL (1), b-1.0E+04copies/mL (2), syncytial virus-1.0E+04copies/mL (3), adenovirus-1.0E+04copies/mL (4), rhinovirus-1.0E+04copies/mL (5), parainfluenza virus-1.0E+04copies/mL (6), respectively, 3 times for each sample simultaneously; the test results are shown in fig. 6.
In summary, the method for detecting the multiple respiratory viruses based on the gene film chip realizes screening of the multiple respiratory viruses in a single respiratory sample by combining multiple PCR and reverse dot hybridization technologies, and can realize automatic screening of the multiple respiratory pathogens in the sample and simultaneously provide a detection result report by combining a full-automatic nucleic acid molecule hybridization instrument and a hybridization reagent system matched with the full-automatic nucleic acid molecule hybridization instrument, and an exemplary detection report template is shown in a specification chart 7, is simple to operate, has high detection sensitivity (IA detection limit is 1.0E+03copies/mL, and the detection limit of other targets is 1.0E+04copies/mL), can complete detection of 1-24 samples within 4 hours, and greatly saves detection reagent cost and labor cost; the UNG enzyme can effectively degrade uracil-containing sequences in PCR products, dUTP and dTTP are mixed according to a certain proportion when a PCR reaction solution is prepared, so that amplification products contain deoxyuracil, and the products are sensitive to the UNG enzyme, so that a newly prepared reaction system can be treated by the UNG enzyme before PCR, and aerosol pollution can be prevented to a certain extent; the specificity and accuracy of the detection result are ensured by taking GAPDH as an internal reference to monitor the nucleic acid extraction and PCR process and taking PC and NC as chip quality control to monitor the hybridization process.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
SEQUENCE LISTING
<110> Sichuan Hua Han Sanzhang biotechnology Co., ltd
<120> Method for detecting various respiratory viruses based on gene film chip
<130> CN113512611A
<140> CN202110647803.6
<141> 2021-06-10
<160> 24
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Claims (4)
1. A method for detecting a plurality of respiratory viruses based on a gene film chip, the method not aiming at obtaining a disease diagnosis result or a health condition, the method comprising: 6 common respiratory viruses are detected simultaneously by combining multiple PCR and reverse dot hybridization technology, GAPDH is used as an internal control to monitor the nucleic acid extraction and PCR amplification process, and PC and NC are used as chip hybridization quality control to monitor the hybridization process;
wherein the common respiratory viruses include influenza a virus, influenza b virus, respiratory syncytial virus, adenovirus, rhinovirus, and parainfluenza virus;
wherein, the forward primer sequence of the influenza A virus is shown as SEQ ID No.4, and the reverse primer sequence is shown as SEQ ID No. 5; the probe sequence is shown as SEQ ID No. 6;
The forward primer sequence of the influenza B virus is shown as SEQ ID No.7, and the reverse primer sequence is shown as SEQ ID No. 8; the probe sequence is shown as SEQ ID No. 9;
The forward primer sequence of the respiratory syncytial virus is shown as SEQ ID No.10, and the reverse primer sequence is shown as SEQ ID No. 11; the probe sequence is shown as SEQ ID No. 12;
the forward primer sequence of adenovirus is shown as SEQ ID No.13, and the reverse primer sequence is shown as SEQ ID No. 14; the probe sequence is shown as SEQ ID No. 15;
The forward primer sequence of the rhinovirus is shown as SEQ ID No.16, and the reverse primer sequence is shown as SEQ ID No. 17; the probe sequence is shown as SEQ ID No. 18;
The forward primer sequence of the parainfluenza virus is shown as SEQ ID No.19, and the reverse primer sequence is shown as SEQ ID No. 20; the probe sequence is shown as SEQ ID No. 21;
the detection method comprises the following detection steps:
(1) Sample pretreatment, specifically: taking a nasal/pharyngeal secretion swab in a preservation tube containing a sample preservation solution, transferring all solutions into a centrifuge tube after vortex shaking, centrifuging at 12000rpm for 5min, and discarding the supernatant;
(2) Nucleic acid extraction, specifically: adding nucleic acid extract and proteinase K into a centrifuge tube, vortex oscillating for 15s, heating in a metal bath with constant temperature of 56 ℃ for 10-15min, adjusting the temperature to 95 ℃ for 2min, centrifuging at 12000rpm for 5min, and taking supernatant as nucleic acid solution participating in PCR reaction;
(3) Multiple PCR sample addition and amplification are specifically as follows: adding components of a PCR amplification system into a centrifuge tube, fully mixing to obtain a multiplex respiratory virus PCR Mix, then adding the multiplex respiratory virus PCR Mix, a nucleic acid solution and double distilled water into a PCR tube to prepare a PCR amplification solution, and finally amplifying the PCR amplification solution according to a designated amplification program to obtain a PCR amplification product;
(4) Membrane chip hybridization, specifically: step ①, adding the PCR amplification product into a membrane chip containing hybridization solution, and heating at 45 ℃ for 30min; step ②, adding hybridization cleaning solution, and heating at 52 ℃ for 3min; step ③, repeating step ② once; step ④, adding an enzyme-labeled solution, and heating at 42 ℃ for 10min; step ⑤, adding an enzyme-labeled cleaning solution I, and heating at 42 ℃ for 3min; step ⑥, adding an enzyme-labeled cleaning solution II, and heating at 37 ℃ for 3min; step ⑦, repeating step ⑥ once; step ⑧, adding a color development liquid, and heating at 37 ℃ for 10min; step ⑨, adding a chromogenic cleaning solution, heating at 37 ℃ for 1min, step ⑩, and repeating step ⑨ twice, wherein the reaction solution is removed by centrifugation after each step in step ①、②、④、⑤、⑥、⑧、⑨ is heated;
(5) And (5) sending out a detection result report.
2. The method for detecting the multiple respiratory viruses based on the gene film chip as claimed in claim 1, wherein the method comprises the following steps: the PCR amplification system comprises UNG enzyme, dATP, dTTP, dCTP, dGTP, dUTP, taq DNA polymerase, reverse transcriptase, PCR buffer solution and primers, wherein the PCR buffer solution is mainly magnesium ion solution, and also comprises potassium ion and betaine; the multiplex PCR system comprises a section of Pos-oligo sequence which is complementary with the PC probe and contains 5' -biotin modification, and is used for hybridization with the PC probe and quality control hybridization process.
3. The method for detecting the multiple respiratory viruses based on the gene film chip as claimed in claim 1, wherein the method comprises the following steps: the influenza a viruses comprise subtype a H1N1-2009, seasonal a flow H1N1, a flow H3N2, a flow H7N9, the influenza b viruses comprise subtype b Yamagata, victoria, the respiratory syncytial viruses comprise subtype respiratory syncytial virus A, B, the adenoviruses comprise subtype 3,4, 7, 55 of human adenovirus and the parainfluenza viruses comprise subtype 1,2,3 of human parainfluenza virus.
4. The method for detecting the multiple respiratory viruses based on the gene film chip as claimed in claim 1, wherein the method comprises the following steps: the composition of the hybridization solution is 2X SSPE,0.1%SDS,30 percent of deionized formamide;
The hybridization rinse consists of 2 XSSPE, 0.5% SDS;
The enzyme-labeled liquid comprises the components of 2X SSPE,0.5%SDS,0.4 mug/mL AP-SA;
the component of the enzyme-labeled cleaning solution I is 2 XSSPE, 0.5% SDS;
The enzyme-labeled cleaning solution II comprises 1M Tris-HCl,5M NaCl,1M MgCl 2;
the color development liquid comprises 0.15mg/mL BCIP,0.30mg/mL NBT,100mmol/L Tris-HCl and 5mmol/L MgCl 2;
The composition of the chromogenic cleaning solution is 1 XPBS.
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