CN114032337A - Respiratory tract pathogen detection kit and preparation method and application thereof - Google Patents
Respiratory tract pathogen detection kit and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a multiple nucleic acid detection kit which can simultaneously detect influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae and parainfluenza viruses I to IV in a single-tube PCR reaction system. The detection kit also provides a respiratory tract pathogen nucleic acid detection method, which can accurately and effectively distinguish and determine each target gene sequence in a sample by a simpler reaction system and lower detection cost, has high detection specificity and sensitivity, is easy to interpret the detection result, and is favorable for large-scale popularization and application in actual detection.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a respiratory tract pathogen detection kit and a preparation method and application thereof.
Background
Respiratory tract pathogen infection is a common clinical infectious disease, and respiratory tract pathogens can be transmitted through air to cause pathogens of acute respiratory tract diseases, have the characteristics of strong infectivity, quick transmission, short latency, acute morbidity and the like, can cause wide acute upper respiratory tract and lower respiratory tract diseases, and seriously harm the health of human beings.
Common respiratory pathogens include influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, mycoplasma pneumoniae, metapneumovirus, rhinovirus and the like. Respiratory infectious diseases have similar clinical symptoms and epidemic characteristics, and early diagnosis, early treatment and early isolation are the basis of prevention. Respiratory tract pathogen detection technologies are various, and main methods include antigen detection, antibody detection, virus isolation and culture, virus nucleic acid detection and the like. However, identification of the type of pathogen infected is difficult through clinical characterization and routine laboratory testing. A large number of documents and patents at home and abroad have reported that the detection of the respiratory viruses by adopting a virus nucleic acid detection method has the obvious advantages of quick detection, simple and convenient operation, good specificity and the like compared with virus culture identification and antigen-antibody detection.
U.S. patent US 6015664 application discloses a nucleic acid detection method using conventional multiplex PCR amplification and linear reverse probe hybridization techniques that can simultaneously detect seven respiratory viruses (influenza a, influenza b, respiratory syncytial a, respiratory syncytial b, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3). However, the traditional multiplex PCR amplification method needs to add a large amount of primer sequences in the same PCR system, which easily generates a large amount of primer dimers, and affects the sensitivity and accuracy of PCR amplification and subsequent hybridization. The linear reverse probe hybridization detection technology also has the limitations of complicated detection operation steps, long detection time consumption, high risk of PCR product pollution in laboratories, and the like.
Poritz et al (PloS one.2011; 6 (10): e26047) describe a nucleic acid detection method based on nested multiplex PCR amplification and microfluidic chip detection technology, which is capable of detecting the genomes of 17 respiratory viruses and 3 respiratory bacteria simultaneously and is named as a FilmArray respiratory syndrome detection kit. The FilmArray detection kit adopts nested PCR technology to improve the sensitivity and specificity of multiplex PCR. Meanwhile, the introduction of the microfluidic chip technology greatly shortens the PCR amplification time. Although the FilmArray detection kit realizes the closed-tube state detection of 20 respiratory pathogens, 102 PCR reaction systems are required to be integrated in the kit, and each PCR reaction system only detects 1 pathogen nucleic acid sequence actually. In addition, the FilmArray detection kit is essentially based on a single PCR detection technology, and has the limitations of low detection flux and high detection cost.
Chinese patent application CN 105936945 a discloses a nucleic acid detection method based on traditional multiplex PCR amplification and four-color real-time fluorescence detection technology, which can simultaneously detect 4 respiratory viruses (influenza virus, respiratory syncytial virus, adenovirus and human metapneumovirus), the real-time fluorescence PCR technology is limited by the number of fluorescence channels detected by a real-time fluorescence quantitative PCR instrument, the method can only detect 4 different viruses in one PCR reaction, and it is difficult to meet the detection requirements of various respiratory viruses.
In review, although various methods of detection of respiratory pathogens have been reported, each has its own limitations. Therefore, there is still a need to develop a more rapid and simple method for detecting respiratory virus nucleic acid with high throughput.
Disclosure of Invention
Based on this, one of the objectives of the present invention is to provide a multiplex nucleic acid detection kit.
The technical scheme is as follows:
a multiple nucleic acid detection kit is characterized by comprising an amplification primer group and a detection probe group aiming at a respiratory tract pathogen nucleic acid sequence, wherein the amplification primer group comprises a CLO primer pair (CLO), and the detection probe group comprises a T probe and a B probe; the CLO primer pair is used for amplifying a respiratory tract pathogen nucleic acid sequence, the T probe comprises a target sequence region and an artificial sequence region, and the target sequence region of the T probe is reversely complementary with the respiratory tract pathogen nucleic acid sequence; the respiratory pathogens are selected from at least one of influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae and parainfluenza virus.
The invention also aims to provide the application of the multiplex nucleic acid detection kit in the detection of respiratory pathogens.
One of the objectives of the present invention is to provide a method for detecting nucleic acid of respiratory pathogens.
The technical scheme is as follows:
obtaining nucleic acid of a biological sample to be detected;
and carrying out PCR detection and melting curve analysis on the biological sample nucleic acid by using the multiplex nucleic acid detection kit.
The invention provides a rapid, simple, sensitive, specific, stable and reliable multiple nucleic acid detection kit based on deep research on respiratory pathogens, and the detection kit can simultaneously detect influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae and parainfluenza virus types I-IV in a single-tube PCR reaction system.
Meanwhile, the invention also provides a respiratory tract pathogen nucleic acid detection method based on the respiratory tract pathogen nucleic acid detection kit prepared by the inventor, the method can accurately and effectively distinguish and measure each target gene sequence in a sample by a simpler reaction system and lower detection cost, the detection specificity is good, the sensitivity is high, the detection result is easy to interpret, and the method is favorable for large-scale popularization and application in actual detection.
Drawings
FIG. 1 is a schematic diagram of the structure of each primer probe in the kit of the present invention.
FIG. 2 is a partial result of the detection of the positive quality control substance by the kit of the present invention in example 1, wherein A is a melting curve under the FAM channel, C is an amplification curve under the FAM channel, B is a melting curve under the VIC channel, and D is an amplification curve under the VIC channel.
FIG. 3 is a partial result of the test of the positive quality control substance by the kit of the present invention in example 1, wherein A is a melting profile under ROX channel, C is an amplification profile under ROX channel, B is a melting profile under CY5 channel, and D is an amplification profile under CY5 channel.
FIG. 4 is a partial result of the sensitivity test of the kit of the present invention using pathogen standards in example 3, wherein A is a melting profile for influenza A virus, D is an amplification profile for influenza A virus, B is a melting profile for influenza B virus, E is an amplification profile for influenza B virus, C is a melting profile for syncytial virus, and F is an amplification profile for syncytial virus.
FIG. 5 shows the results of the sensitivity detection part of the kit of the present invention using the pathogen standards in example 3, wherein A is a melting profile for adenovirus, D is an amplification profile for adenovirus, B is a melting profile for parainfluenza virus, E is an amplification profile for parainfluenza virus, C is a melting profile for Mycoplasma pneumoniae, and F is an amplification profile for Mycoplasma pneumoniae.
FIG. 6 shows the results of the detection of a portion of the sensitivity of the kit of the present invention using the pathogen standards in example 3, wherein A is a melting profile for metapneumovirus, C is an amplification profile for metapneumovirus, B is a melting profile for rhinovirus, and D is an amplification profile for rhinovirus.
FIG. 7 shows the results of the specific detection test group of the kit of the present invention in example 4, wherein A is the melting profile of 24 cross-reactive-species-confirmed pathogens and B is the amplification profile of 24 cross-reactive-species-confirmed pathogens.
FIG. 8 shows the results of the specific detection control group of the kit of the present invention in example 4, wherein A is a melting profile for influenza A virus, Mycoplasma pneumoniae, adenovirus and internal standard in the sample, and B is an amplification profile for influenza A virus, Mycoplasma pneumoniae, adenovirus and internal standard in the sample.
FIG. 9 is a graph showing the results of partially detecting a sample containing an interfering substance with the kit of the present invention in example 5, wherein A is a melting profile for influenza A virus in the sample, C is an amplification profile for influenza A virus in the sample, B is a melting profile for adenovirus in the sample, and D is an amplification profile for adenovirus in the sample.
FIG. 10 is a graph showing the results of partial detection of a sample containing an interfering substance by the kit of the present invention in example 5, wherein A is a melting profile for Mycoplasma pneumoniae in the sample, C is an amplification profile for Mycoplasma pneumoniae in the sample, B is a melting profile for influenza B virus in the sample, and D is an amplification profile for influenza B virus in the sample.
FIG. 11 is a graph showing the results of partially detecting a sample containing an interfering substance with the kit of the present invention in example 5, wherein A is a melting profile for metapneumovirus in the sample, C is an amplification profile for metapneumovirus in the sample, B is a melting profile for parainfluenza virus in the sample, and D is an amplification profile for parainfluenza virus in the sample.
FIG. 12 is a graph showing the results of the partial detection of a sample containing an interfering substance by the kit of the present invention in example 5, wherein A is a melting profile for respiratory syncytial virus in the sample, C is an amplification profile for respiratory syncytial virus in the sample, B is a melting profile for rhinovirus in the sample, and D is an amplification profile for rhinovirus in the sample.
FIG. 13 shows the partial detection results of primer optimization in the kit of the invention in example 6, wherein A is a melting curve for influenza A virus, D is an amplification curve for influenza A virus, B is a melting curve for influenza B virus, E is an amplification curve for influenza B virus, C is a melting curve for syncytial virus, and F is an amplification curve for syncytial virus.
Fig. 14 is a partial detection result of primer optimization in the kit of the invention in example 6, in which a is a melting profile for an internal reference HBB gene, D is an amplification profile for an internal reference HBB gene, B is a melting profile for rhinovirus, E is an amplification profile for rhinovirus, C is a melting profile for mycoplasma pneumoniae, and F is an amplification profile for mycoplasma pneumoniae.
FIG. 15 shows the results of the primer optimization of the kit of the invention in example 6, wherein A is a melting profile for adenovirus, D is an amplification profile for adenovirus, B is a melting profile for parainfluenza virus type I, E is an amplification profile for parainfluenza virus type I, C is a melting profile for parainfluenza virus type II, and F is an amplification profile for parainfluenza virus type II.
FIG. 16 shows the partial detection results of primer optimization in the kit of the present invention in example 6, wherein A is a melting profile for parainfluenza virus type III, D is an amplification profile for parainfluenza virus type III, B is a melting profile for parainfluenza virus type IV, E is an amplification profile for parainfluenza virus type IV, C is a melting profile for human metapneumovirus, and F is an amplification profile for human metapneumovirus.
Detailed Description
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or," unless the context clearly dictates otherwise.
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The present invention will be described in further detail with reference to specific examples.
Some embodiments of the present invention provide a multiplex nucleic acid detection kit, wherein the multiplex nucleic acid detection kit comprises an amplification primer set and a detection probe set for a respiratory tract pathogen nucleic acid sequence, the amplification primer set comprises a CLO primer pair, and the detection probe set comprises a T probe and a B probe; the CLO primer pair is used for amplifying a respiratory tract pathogen nucleic acid sequence, the T probe comprises a target sequence region and an artificial sequence region, and the target sequence region is reversely complementary to the respiratory tract pathogen nucleic acid sequence; the respiratory pathogens are selected from at least one of influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae and parainfluenza virus.
In some embodiments, the multiplex nucleic acid detection kit comprises at least one set of any of the following components:
a first group: the kit comprises a CLO primer pair, a T probe and a B probe aiming at influenza A virus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 1-SEQ ID NO.2, the sequence of the T probe is shown as SEQ ID NO.27, and the sequence of the B probe is shown as SEQ ID NO. 28;
second group: the kit comprises a CLO primer pair, a T probe and a B probe aiming at influenza B virus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 3-SEQ ID NO.4, the sequence of the T probe is shown as SEQ ID NO.29, and the sequence of the B probe is shown as SEQ ID NO. 30;
third group: the kit comprises a CLO primer pair, a T probe and a B probe aiming at the respiratory syncytial virus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 5-SEQ ID NO.6, the sequence of the T probe is shown as SEQ ID NO.31, and the sequence of the B probe is shown as SEQ ID NO. 32;
and a fourth group: CLO primer pair, T probe and B probe aiming at adenovirus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 7-SEQ ID NO.8, the sequence of the T probe is shown as SEQ ID NO.33, and the sequence of the B probe is shown as SEQ ID NO. 34;
and a fifth group: the probe comprises a CLO primer pair, a T probe and a B probe aiming at mycoplasma pneumoniae, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 9-SEQ ID NO.10, the sequence of the T probe is shown as SEQ ID NO.35, and the sequence of the B probe is shown as SEQ ID NO. 36;
a sixth group: the probe comprises a CLO primer pair, a T probe and a B probe aiming at parainfluenza virus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 11-SEQ ID NO.18, the sequence of the T probe is shown as SEQ ID NO. 37-SEQ ID NO.40, and the sequence of the B probe is shown as SEQ ID NO. 41;
a seventh group: CLO primer pairs, T probes and B probes aiming at rhinoviruses, wherein the sequences of the CLO primer pairs are shown as SEQ ID NO. 19-SEQ ID NO.20, the sequences of the T probes are shown as SEQ ID NO.42, and the sequences of the B probes are shown as SEQ ID NO. 43;
and an eighth group: the CLO probe comprises a CLO primer pair, a T probe and a B probe aiming at human metapneumovirus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 21-SEQ ID NO.22, the sequence of the T probe is shown as SEQ ID NO.44, and the sequence of the B probe is shown as SEQ ID NO. 45.
It should be noted that the above-mentioned "first", "second", "third" and "fourth" are used for descriptive purposes only and are used for distinguishing defined substances, and are not used to define an order or primary or secondary in any way.
In some embodiments, the multiplex nucleic acid detection kit comprises any of the above at least three sets of components; preferably at least five of the above components; more preferably, the above eight components are included.
In some embodiments, the multiplex nucleic acid detection kit further comprises a universal primer pair, wherein an upstream universal primer in the universal primer pair is identical to a loop region of an upstream CLO primer in the CLO primer pair; and the downstream universal primer in the universal primer pair is consistent with the loop region of the downstream CLO primer in the CLO primer pair. Further, the sequence of the universal primer pair is shown as SEQ ID NO. 25-SEQ ID NO. 26.
In some embodiments, the universal primers can amplify all PCR products generated from the first 2 cycles after the 3 rd cycle of PCR amplification, thereby ensuring amplification uniformity of each target.
In some embodiments, the multiplex nucleic acid detection kit further comprises an amplification primer set and a detection probe set for the internal reference nucleic acid sequence; further, the internal reference is an HBB gene.
In some embodiments, the multiplex nucleic acid detection kit further comprises the following components:
ninth group: the kit comprises a CLO primer pair, a T probe and a B probe aiming at an HBB gene, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 23-SEQ ID NO.24, the sequence of the T probe is shown as SEQ ID NO.46, and the sequence of the B probe is shown as SEQ ID NO. 47.
Some embodiments of the invention provide the use of the multiplex nucleic acid detection kit described above in the detection of respiratory pathogens.
In some embodiments, the respiratory pathogens to be detected by typing with the kit include at least one of influenza a virus, influenza b virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, parainfluenza virus.
Some embodiments of the invention provide a method for detecting a respiratory pathogen nucleic acid, comprising the steps of:
obtaining nucleic acid of a biological sample to be detected;
and carrying out PCR detection and melting curve analysis on the biological sample nucleic acid by using the multiplex nucleic acid detection kit.
In some embodiments, the type of the target nucleic acid sequence of the respiratory pathogen present in the reaction system is determined by melting curve analysis of the PCR reaction product.
In some embodiments, the reaction sequence for the PCR detection is touchdown PCR.
In some embodiments, the biological sample includes, but is not limited to, serum, plasma, whole blood, sputum, swabs, lavage, fresh tissue, formalin-fixed paraffin-embedded tissue, urine, bacterial cultures, viral cultures, cell line cultures, and synthetic plasmids.
In some embodiments, when the nucleic acid of the biological sample is ribonucleic acid, the reaction system further comprises reverse transcriptase, and the reaction procedure further comprises a reverse transcription PCR procedure.
EXAMPLE 1 composition of nucleic acid detection kit for respiratory pathogens
1. Primers and probes
The respiratory tract pathogen nucleic acid detection kit comprises an amplification primer group and a detection probe group aiming at a nucleic acid sequence of a respiratory tract pathogen, wherein the amplification primers in the kit are specifically shown in the following tables 1-1 (the 'F' represents an upstream primer, and the 'R' represents a downstream primer), and the probes aiming at the respiratory tract pathogens are specifically shown in the following tables 1-2:
TABLE 1-1
Tables 1 to 2
The "-" linkage in the table indicates the position of the modifying group and the name of the modifying group.
2. Quality control product
The kit contains a negative quality control product and a positive quality control product, and the negative and positive quality control products and a sample to be detected need to be synchronously processed. The positive quality control product consists of pseudoviruses containing influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, parainfluenza virus and internal reference gene segments; the negative quality control product consists of pseudovirus containing reference gene segments. The negative and positive quality control products were purchased from Bai' ao (Suzhou) Biotechnology Ltd.
3. Enzyme concentration
The concentration of reverse transcriptase is 5U/. mu.L-15U/. mu.L, and the reverse transcriptase can be murine leukemia reverse transcriptase (MMLV) or Tth enzyme; the DNA polymerase is 5U/. mu.L-15U/. mu.L, and the DNA polymerase can be Taq enzyme.
4. PCR system
4.1 primer preparation: CLO-F1/CLO-R1-CLO-F12/CLO-R12 and Up-F, Up-R, for 26 strips; centrifuging at 10,000rpm for 3 min; dissolve with TE to 100 pmol/. mu.L of the mother liquor, prepare premixes according to the following tables 1-3:
tables 1 to 3
100 μ L of the premix prepared in the above table had a final single CLO-F1/CLO-R1-CLO-F12/CLO-R12 concentration of 1.6 pmol/. mu.L and a final single Up-F, Up-R concentration of 16 pmol/. mu.L, and was designated HXD-T.
4.2 preparing a probe: 21T 1/B1-T11/B12 in total; centrifuging at 10000rpm for 3 min; dissolve with TE to 100 pmol/. mu.L of the mother liquor, prepare premixes according to the following tables 1-4:
tables 1 to 4
| Serial number | Components | Add volume (μ L) |
| 1 | 12 pieces of T1-T12 | 1.6 |
| 2 | B1-B12, 9 strips | 0.8 |
| 3 | TE solution | 73.6 |
The 100. mu.L premix prepared in the above manner had a final concentration of 1.6pmol/μ L, B1-B12 for each probe T1-T12 and 0.8pmol/μ L for each probe, and the premix was designated HXD-B.
4.3PCR reaction system preparation, the PCR reaction system is prepared according to the following tables 1-5:
tables 1-5 information tables for PCR reaction System preparation
After the reaction system is prepared, the sample to be detected, the negative control and the positive control are added by 5 mu L, evenly mixed by oscillation and are arranged on a machine after centrifugation.
5. PCR reaction procedure
The PCR reaction program was set up as follows using a touchdown PCR program, with the annealing temperature being reduced by 1 ℃ for each 1 cycle increase, for the first 6 cycles. The specific reaction procedures are shown in tables 1-6 below:
tables 1 to 6
6. Analysis of results
The Tm values for the 8 respiratory pathogens in each channel are shown in tables 1-7 below:
tables 1 to 7
Positive control interpretation: melting peaks are found in FAM, VIC, ROX and CY5 channels, which respectively correspond to the melting peaks of influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae, parainfluenza virus and internal reference genes, and the positive quality control products are judged to be qualified. If one or more of the melting peaks do not exist, judging that the reagent is invalid; and if the melting peak except the pathogen appears, judging that the pollution exists in the current detection.
Wherein the detection results of qualified positive quality control products adopting the kit are shown in figures 2-3.
Negative control interpretation: no melting peak exists in FAM, VIC and ROX channels, a melting peak exists in CY5 channel, which corresponds to the melting peak of the internal reference gene, and the negative quality control product is judged to be qualified. If the FAM, VIC and ROX channels have melting peaks, judging that the current detection has pollution.
Judging a sample to be detected:
1) in FAM, VIC and ROX channels, when a melting peak exists in a specific pathogen Tm reference value range, the pathogen is judged to be positive;
2) when two or more melting peaks appear at the same time, judging that the sample is infected with two or more pathogens at the same time;
3) when FAM, VIC and ROX channels have no melting peak and CY5 channel has a melting peak, judging that the sample has no pathogen infection in the detection range;
4) if no melting peak exists in FAM, VIC, ROX and CY5 channels, the sample is judged to be invalid, and resampling or nucleic acid re-extraction and then detection are recommended.
For example, some of the test results are shown in tables 1-8 below, and the test results are analyzed as follows:
tables 1 to 8
Note: + represents the presence of a melting peak, -represents the absence of a melting peak, ± represents the presence or absence of a melting peak, and/represents the absence of a detection target at the Tm value.
Example 2 detection of clinical samples by the respiratory tract pathogen nucleic acid detection kit of the present invention
By using the respiratory tract pathogen nucleic acid detection kit, collected throat swab clinical specimens are subjected to nucleic acid extraction and then detected according to the method steps described in the embodiment 1, and the detection results are counted and shown in the following tables 2-1-2-9. The control method is detection of 13 respiratory tract pathogen multiple detection kits (PCR capillary electrophoresis fragment analysis method) (national mechanical standard 20183400518).
TABLE 2-1 statistical table of negative and positive influenza A virus
TABLE 2-2 statistical tables for negative and positive influenza B virus
TABLE 2-3 statistical tables for negative and positive respiratory syncytial virus
TABLE 2-4 statistical tables for negative and positive adenovirus
TABLE 2-5 statistical tables of negative and positive parainfluenza Virus
TABLE 2-6 Mycoplasma pneumoniae negative and positive statistical tables
TABLE 2-7 statistics of yin-positivity of metapneumovirus
TABLE 2-8 statistics of negative and positive rhinovirus
Tables 2-9 statistics of negative and positive for the population samples
The statistical data show that the respiratory tract pathogen nucleic acid detection kit has good detection performance for clinical influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae and parainfluenza virus samples.
Example 3 sensitive detection of the kit of the invention
The detection sensitivity of the kit of the present invention was analyzed by detecting standards (purchased from biosciences, inc. of bond, guangzhou) having different concentration gradients according to the procedure of example 1. The detection results are shown in fig. 4-6, and after further analysis, the detection limits of the kit of the invention on various pathogens are statistically obtained as shown in the following table 3-1:
TABLE 3-1
| Pathogens | Detection limit | Pathogens | Detection limit |
| Influenza A virus (IFVA) | 2.0TCID50/mL | Rhinovirus (RhV) | 500copies/mL |
| Influenza B virus (IFVB) | 2.0TCID50/mL | Parainfluenza virus (PIV) | 500copies/mL |
| Respiratory Syncytial Virus (RSV) | 500copies/mL | Human metapneumovirus (hMPV) | 500copies/mL |
| Adenovirus (Adv) | 500copies/mL | Mycoplasma Pneumoniae (MP) | 500copies/mL |
Example 4 specific detection of the kit of the invention
The kit is used for detecting common respiratory pathogens which are likely to generate cross reaction, an experimental group and a control group are arranged, the experimental group is formed by respectively adding the respiratory pathogens in the following table 4-1 into 24 pharyngeal swab clinical samples (negative in the detection range of the kit) to serve as samples to be detected, and the control group is a mixed clinical sample with 8 respiratory viruses positive in the detection range of the kit. The detection results are shown in FIGS. 7-8, which show that the detection results of the experimental group are all negative, and the detection results of the control group are all normal, and show that the 24 respiratory pathogens in the table 4-1 do not have cross reaction with the kit.
TABLE 4-1 Cross-reactivity verification experiment group calling inhalant pathogen (copies/mL)
Example 5 Effect of interfering substances on the kits of the invention
Preparing an experimental group sample and a control group sample, wherein the clinical samples are respectively added with a certain proportion of treatment medicines as the experimental group, and the samples without the addition of medicines are the control group, such as common anti-cold medicines, glucocorticoid and antibiotics, and the detection of the kit is not influenced. Wherein a composition comprising phenylephrine (100. mu.g/mL), oxymetazoline (100. mu.g/mL), beclomethasone (50. mu.g/mL), dexamethasone (100. mu.g/mL), flunisolide (10. mu.g/mL), triamcinolone acetonide (100. mu.g/mL), budesonide (50. mu.g/mL), mometasone (50. mu.g/mL), fluticasone (50. mu.g/mL), histamine hydrochloride (50. mu.g/mL), intranasal live influenza virus vaccine (30. mu.g/mL), benzocaine (50. mu.g/mL), menthol (32. mu.g/mL), zanamivir (100. mu.g/mL), ribavirin (100. mu.g/mL), oseltamivir (100. mu.g/mL), peramivir (100. mu.g/mL), mupirocin (100. mu.g/mL) was detected, Samples of tobramycin (50. mu.g/mL).
The detection results of the experimental group and the control group of each clinical sample are shown in fig. 9-12, and the results show that the interference substances have no influence on the detection results and do not influence the accuracy of the kit.
Example 6 optimization and combinatorial validation of primer probes
The primer probe in the amplification reaction system may form a dimer between the primer and (or) the probe based on the base complementary pairing principle, and particularly, a primer dimer is easily generated when a complementary pairing base exists at the 3' end. The conservation of the target gene section and the reduction of the mutual interference between different primer probes are ensured at the beginning of the design, and the primer probes need to be designed and screened elaborately.
In the invention, the inventor designs a plurality of pairs of primers for comparison research at the same time, as shown in the following tables 6-1 to 6-8, and simultaneously detects the same sample, and as shown in the following tables 13 to 16, selects a primer pair with higher amplification efficiency as a preferred primer, thereby better amplifying each target pathogen.
TABLE 6-1 influenza A primers
TABLE 6-2 adenovirus primer sets
TABLE 6-3 Mycoplasma pneumoniae primers
TABLE 6-4 primers for influenza B virus
TABLE 6-5 Metapneumovirus primers
TABLE 6-6 parainfluenza Virus primers
TABLE 6-7 respiratory syncytial virus primers
TABLE 6-8 rhinovirus primers
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Guangzhou City gold boundary Rui Biotechnology Limited liability company
<120> respiratory tract pathogen detection kit, preparation method and application thereof
<160> 69
<170> SIPOSequenceListing 1.0
<210> 1
<211> 58
<212> DNA
<213> Artificial Sequence
<400> 1
attttggaca aagcgtctac gctgcatgta gtcatcactg agtcatcgcc tcgctcac 58
<210> 2
<211> 60
<212> DNA
<213> Artificial Sequence
<400> 2
acacagatct tgaggctttc atggaatcag cagagacggc aacttatata aagacaagac 60
<210> 3
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 3
atcaggaaat gtagtcatca ctgagtcatc ggaacaacag c 41
<210> 4
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 4
gctgagctca gcagagacgg caacttataa tggccttc 38
<210> 5
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 5
aaacagacat aagcagctca gtaaatgtag tcatcactga gtcatcgctt ctctaggag 59
<210> 6
<211> 58
<212> DNA
<213> Artificial Sequence
<400> 6
ttcattgact tgagatattg atgcatccag cagagacggc aacttatact catcagaa 58
<210> 7
<211> 51
<212> DNA
<213> Artificial Sequence
<400> 7
cgttgccggc cgagaaggat gtagtcatca ctgagtcatc gtgcgcaggt a 51
<210> 8
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 8
gacttttgag gtggatccca tggacagcag agacggcaac ttatagccca ccctt 55
<210> 9
<211> 58
<212> DNA
<213> Artificial Sequence
<400> 9
aatacatcta cccttaccgt tacagatgta gtcatcactg agtcatcgca tgtgagct 58
<210> 10
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 10
ctaccgttaa cttctggtta aagcgctcca gcagagacgg caacttatac tcgttagca 59
<210> 11
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 11
tgtatatcaa ctgtgttcaa ctccatgtag tcatcactga gtcatcggtt gatgaaaga 59
<210> 12
<211> 44
<212> DNA
<213> Artificial Sequence
<400> 12
cttgtctttt cttcagcaga gacggcaact tataccaagt tatc 44
<210> 13
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 13
ttctgcagct atatgtagtc atcactgagt catcgtaatc acatc 45
<210> 14
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 14
ggacatagtt cagcagagac ggcaacttat acgagcatct g 41
<210> 15
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 15
tgtatatcaa ctgtgttcaa ctccatgtag tcatcactga gtcatcggtt gatgaaaga 59
<210> 16
<211> 60
<212> DNA
<213> Artificial Sequence
<400> 16
ttgcctttgt agtatattcc tggtccacag cagagacggc aacttataga tgggtataat 60
<210> 17
<211> 48
<212> DNA
<213> Artificial Sequence
<400> 17
tagattatcc caattatgta gtcatcactg agtcatcgtt ccaactgc 48
<210> 18
<211> 42
<212> DNA
<213> Artificial Sequence
<400> 18
tcgactttaa acagcagaga cggcaactta taaagggtca cc 42
<210> 19
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 19
caatataagg aataaaaaga aacacggatg tagtcatcac tgagtcatcg cccaaagta 59
<210> 20
<211> 56
<212> DNA
<213> Artificial Sequence
<400> 20
agacctgcat gtgcttgatt gtgagcagca gagacggcaa cttatatccg gcccct 56
<210> 21
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 21
gggtggtatt gtaaaaatgc aggatgtagt catcactgag tcatcgcact gtttactac 59
<210> 22
<211> 56
<212> DNA
<213> Artificial Sequence
<400> 22
aagccaccaa agcaccgaga ggcagcagag acggcaactt atatagtgca accatg 56
<210> 23
<211> 42
<212> DNA
<213> Artificial Sequence
<400> 23
gtggctcgaa tgtagtcatc actgagtcat cgtctgctca ct 42
<210> 24
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 24
ggctgaggca gcagagacgg caacttatag gagaatgg 38
<210> 25
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 25
atgtagtcat cactgagtca tcg 23
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 26
cagcagagac ggcaacttat a 21
<210> 27
<211> 48
<212> DNA
<213> Artificial Sequence
<400> 27
tacggcgacc accgagatat atgttcacgc tcaccgtgcc cagtcgcg 48
<210> 28
<211> 63
<212> DNA
<213> Artificial Sequence
<400> 28
ggggatgttc tctattttgt attcttcatc tttcatatat ctcggtggtc gccgtaagaa 60
cat 63
<210> 29
<211> 57
<212> DNA
<213> Artificial Sequence
<400> 29
ggcgaccacc tacacctgaa catcaaatgc ttcatgaaag ctcacacatc ttccgcg 57
<210> 30
<211> 66
<212> DNA
<213> Artificial Sequence
<400> 30
ggggaagagt tctattctgt atgcgtacac gctttgatgt tcaggtgtag gtggtcgcca 60
<210> 31
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 31
gcgaccacct agatcacact atgacatgtt ccaatcatcc atgccagcag acgcg 55
<210> 32
<211> 75
<212> DNA
<213> Artificial Sequence
<400> 32
ggggcggcct acgtgcgatc ggccgtccgg ctggccagct cttgtcatag tgtgatctag 60
<210> 33
<211> 48
<212> DNA
<213> Artificial Sequence
<400> 33
tgactacatg tctctatcga agtctttgac gtggtccgtg tgcacgcg 48
<210> 34
<211> 67
<212> DNA
<213> Artificial Sequence
<400> 34
ggggtcggcg cctccgtcac gctgcactgc acattctggc tcgatagaga catgtagtca 60
<210> 35
<211> 47
<212> DNA
<213> Artificial Sequence
<400> 35
agctgacaag gttcatgacg accattacca tgggtgatac cgccgcg 47
<210> 36
<211> 58
<212> DNA
<213> Artificial Sequence
<400> 36
ggggtattac ggagacggtg ctccgtgtgg tcgtcatgaa ccttgtcagc tcgtaata 58
<210> 37
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 37
gtcttgaagt cctgtgggat tcccaatgtt aatcagagtg tttgcaatga tcgcg 55
<210> 38
<211> 54
<212> DNA
<213> Artificial Sequence
<400> 38
gtcttgaagt cctgtgggat cattcaccag aagccagcat agatagagta cgcg 54
<210> 39
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 39
gtcttgaagt cctgtgggat gcatcatcag gcatagaaga tattgtactt gcgcg 55
<210> 40
<211> 58
<212> DNA
<213> Artificial Sequence
<400> 40
gcggagatag acataactga gaattccatc attctcttta ggtcaaaccc attgcgcg 58
<210> 41
<211> 77
<212> DNA
<213> Artificial Sequence
<400> 41
ggggctacgt ggtaagtgtt cagtcacgct cctttcgacc tgcgccgaat cccacaggac 60
ttcaagacaa cacgtag 77
<210> 42
<211> 52
<212> DNA
<213> Artificial Sequence
<400> 42
tgttagagtg cgagtcgtca atatagaatg cggctaacct taaccccgcg cg 52
<210> 43
<211> 68
<212> DNA
<213> Artificial Sequence
<400> 43
ggggggacgg atgtcttgat agttatagcc taattctata ttgacgactc gcactctaac 60
<210> 44
<211> 56
<212> DNA
<213> Artificial Sequence
<400> 44
gccagtgaac tggcagaccg tcctgcgaca cagcagcagg aattaatgtt gccgcg 56
<210> 45
<211> 76
<212> DNA
<213> Artificial Sequence
<400> 45
ggggggccga gctccgcctg cgtcggagct acgccaactc ctttcaggac ggtctgccag 60
<210> 46
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 46
ctacacgaca ctcttcctac aagctccgcc tcctgggttc acgcg 45
<210> 47
<211> 66
<212> DNA
<213> Artificial Sequence
<400> 47
gggggtgaca tcgtgatact tctctactgt ctttttactg taggaagagt gtcgtgtaga 60
<210> 48
<211> 54
<212> DNA
<213> Artificial Sequence
<400> 48
attagaacag caatacaaca aatgtagtca tcactgagtc atcggtattg gaac 54
<210> 49
<211> 52
<212> DNA
<213> Artificial Sequence
<400> 49
cgacctctta tattatggaa tccagcagag acggcaactt atacagaggt at 52
<210> 50
<211> 54
<212> DNA
<213> Artificial Sequence
<400> 50
ttgtgaccta agagtagtat atgtagtcat cactgagtca tcgatagacg ctat 54
<210> 51
<211> 52
<212> DNA
<213> Artificial Sequence
<400> 51
tgacatctat gacaccttca gcagagacgg caacttatat tctgtgtatg gt 52
<210> 52
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 52
ctacgatgaa ctaagattga tgtatgtagt catcactgag tcatcggacc gtcccatac 59
<210> 53
<211> 56
<212> DNA
<213> Artificial Sequence
<400> 53
cacactatat ctataccatc taacagcaga gacggcaact tataaatagt gaactg 56
<210> 54
<211> 52
<212> DNA
<213> Artificial Sequence
<400> 54
agacaactat acacaacatt atgtagtcat cactgagtca tcgtgtaaca cg 52
<210> 55
<211> 51
<212> DNA
<213> Artificial Sequence
<400> 55
tagataagaa ccgcaaacca gcagagacgg caacttatag aaatccattt g 51
<210> 56
<211> 60
<212> DNA
<213> Artificial Sequence
<400> 56
gtagaagaaa gcatccataa tgcaggatgt agtcatcact gagtcatcgg tctgaaggtt 60
<210> 57
<211> 57
<212> DNA
<213> Artificial Sequence
<400> 57
tctataccat ctatattcac ttatcagcag agacggcaac ttatagcgga gtgaggt 57
<210> 58
<211> 58
<212> DNA
<213> Artificial Sequence
<400> 58
attatgtgaa gccttgaaca actccatgta gtcatcactg agtcatcggt ggctctat 58
<210> 59
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 59
cgtctatact ctctaattct tgcagcagag acggcaactt atatagcatt taccg 55
<210> 60
<211> 56
<212> DNA
<213> Artificial Sequence
<400> 60
gccattatgt cctgaagaaa tgtagtcatc actgagtcat cggtctccaa cactct 56
<210> 61
<211> 51
<212> DNA
<213> Artificial Sequence
<400> 61
ctatgtgaag ttgtgaatcc agcagagacg gcaacttata aatctcacgc t 51
<210> 62
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 62
cgtctctata cactaatctt actccatgta gtcatcactg agtcatcgca gcgtcagag 59
<210> 63
<211> 57
<212> DNA
<213> Artificial Sequence
<400> 63
ccacaattca acaatccatg gtccacagca gagacggcaa cttataaata gtgaact 57
<210> 64
<211> 52
<212> DNA
<213> Artificial Sequence
<400> 64
gcaatataca catgataaga tgtagtcatc actgagtcat cggacgcctg ca 52
<210> 65
<211> 51
<212> DNA
<213> Artificial Sequence
<400> 65
gcagcgatct gaggtataac agcagagacg gcaacttata tatagcgaat a 51
<210> 66
<211> 62
<212> DNA
<213> Artificial Sequence
<400> 66
tattactcgg actcggtgta tcagtaaatg tagtcatcac tgagtcatcg attcagccgc 60
<210> 67
<211> 60
<212> DNA
<213> Artificial Sequence
<400> 67
tcgtttgcta tttaggtgtc tatgcatcca gcagagacgg caacttatag tctgaaaccg 60
<210> 68
<211> 63
<212> DNA
<213> Artificial Sequence
<400> 68
aacgaataca tcactacaag aaacacggat gtagtcatca ctgagtcatc gctgggcttc 60
ctt 63
<210> 69
<211> 67
<212> DNA
<213> Artificial Sequence
<400> 69
atatgctata cagtctctat acacttgtga gcagcagaga cggcaactta tataaagatt 60
Claims (10)
1. A multiplex nucleic acid detection kit is characterized by comprising an amplification primer group and a detection probe group aiming at a nucleic acid sequence of a respiratory pathogen, wherein the amplification primer group comprises a CLO primer pair, and the detection probe group comprises a T probe and a B probe;
the CLO primer pair is used for amplifying a respiratory tract pathogen nucleic acid sequence, the T probe and the B probe both comprise a target sequence region and an artificial sequence region, and the target sequence region is reversely complementary with the respiratory tract pathogen nucleic acid sequence;
the respiratory pathogens are selected from at least one of influenza A virus, influenza B virus, respiratory syncytial virus, rhinovirus, adenovirus, human metapneumovirus, mycoplasma pneumoniae and parainfluenza virus.
2. The multiplex nucleic acid detection kit according to claim 1, comprising at least one set of any of the following components:
a first group: the kit comprises a CLO primer pair, a T probe and a B probe aiming at influenza A virus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 1-SEQ ID NO.2, the sequence of the T probe is shown as SEQ ID NO.27, and the sequence of the B probe is shown as SEQ ID NO. 28;
second group: the kit comprises a CLO primer pair, a T probe and a B probe aiming at influenza B virus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 3-SEQ ID NO.4, the sequence of the T probe is shown as SEQ ID NO.29, and the sequence of the B probe is shown as SEQ ID NO. 30;
third group: the kit comprises a CLO primer pair, a T probe and a B probe aiming at the respiratory syncytial virus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 5-SEQ ID NO.6, the sequence of the T probe is shown as SEQ ID NO.31, and the sequence of the B probe is shown as SEQ ID NO. 32;
and a fourth group: CLO primer pair, T probe and B probe aiming at adenovirus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 7-SEQ ID NO.8, the sequence of the T probe is shown as SEQ ID NO.33, and the sequence of the B probe is shown as SEQ ID NO. 34;
and a fifth group: the probe comprises a CLO primer pair, a T probe and a B probe aiming at mycoplasma pneumoniae, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 9-SEQ ID NO.10, the sequence of the T probe is shown as SEQ ID NO.35, and the sequence of the B probe is shown as SEQ ID NO. 36;
a sixth group: the probe comprises a CLO primer pair, a T probe and a B probe aiming at parainfluenza virus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 11-SEQ ID NO.18, the sequence of the T probe is shown as SEQ ID NO. 37-SEQ ID NO.40, and the sequence of the B probe is shown as SEQ ID NO. 41;
a seventh group: CLO primer pairs, T probes and B probes aiming at rhinoviruses, wherein the sequences of the CLO primer pairs are shown as SEQ ID NO. 19-SEQ ID NO.20, the sequences of the T probes are shown as SEQ ID NO.42, and the sequences of the B probes are shown as SEQ ID NO. 43;
and an eighth group: the CLO probe comprises a CLO primer pair, a T probe and a B probe aiming at human metapneumovirus, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 21-SEQ ID NO.22, the sequence of the T probe is shown as SEQ ID NO.44, and the sequence of the B probe is shown as SEQ ID NO. 45.
3. The multiple nucleic acid detection kit according to any one of claims 1 to 2, further comprising a universal primer pair, and further wherein the sequence of the universal primer pair is shown in SEQ ID No.25 to SEQ ID No. 26.
4. The multiplex nucleic acid detection kit according to any one of claims 1 to 2, further comprising an amplification primer set and a detection probe set for an internal reference nucleic acid sequence; further, the internal reference is an HBB gene.
5. The multiplex nucleic acid detection kit according to claim 4, further comprising the following components:
ninth group: the kit comprises a CLO primer pair, a T probe and a B probe aiming at an HBB gene, wherein the sequence of the CLO primer pair is shown as SEQ ID NO. 23-SEQ ID NO.24, the sequence of the T probe is shown as SEQ ID NO.46, and the sequence of the B probe is shown as SEQ ID NO. 47.
6. Use of the multiplex nucleic acid detection kit according to any one of claims 1 to 5 for the detection of respiratory pathogens.
7. A method for detecting a nucleic acid of a respiratory pathogen, comprising the steps of:
obtaining nucleic acid of a biological sample to be detected;
performing PCR detection and melting curve analysis on the biological sample nucleic acid by using the multiplex nucleic acid detection kit according to any one of claims 1 to 5.
8. The method of claim 7, wherein the reaction program of the PCR detection is touchdown PCR.
9. The multiplex nucleic acid detection method of any one of claims 7 to 8, wherein the biological sample includes, but is not limited to, serum, plasma, whole blood, sputum, swab, lavage fluid, fresh tissue, formalin-fixed paraffin-embedded tissue, urine, bacterial culture, viral culture, cell line culture, and artificially synthesized plasmid.
10. The multiplex nucleic acid detection method of any one of claims 7 to 9, wherein when the nucleic acid of the biological sample is ribonucleic acid, the reaction system further comprises reverse transcriptase, and the reaction procedure further comprises reverse transcription PCR.
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| CN202111530572.7A CN114032337A (en) | 2021-12-14 | 2021-12-14 | Respiratory tract pathogen detection kit and preparation method and application thereof |
| PCT/CN2022/097194 WO2023109031A1 (en) | 2021-12-14 | 2022-06-06 | Respiratory pathogen detection kit, and preparation method therefor and use thereof |
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| CN202111530572.7A CN114032337A (en) | 2021-12-14 | 2021-12-14 | Respiratory tract pathogen detection kit and preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023109031A1 (en) * | 2021-12-14 | 2023-06-22 | 广州市金圻睿生物科技有限责任公司 | Respiratory pathogen detection kit, and preparation method therefor and use thereof |
| CN117867180A (en) * | 2024-03-12 | 2024-04-12 | 北京雅康博生物科技有限公司 | Primer combination, kit and application for detecting respiratory tract pathogens |
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| CN117867180A (en) * | 2024-03-12 | 2024-04-12 | 北京雅康博生物科技有限公司 | Primer combination, kit and application for detecting respiratory tract pathogens |
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| WO2023109031A1 (en) | 2023-06-22 |
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