CN117568524A - Primer probe group for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP by multiplex PCR, application and kit - Google Patents
Primer probe group for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP by multiplex PCR, application and kit Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to a primer probe group, application and a kit for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP by multiplex PCR, which comprise primers and probes with nucleotide sequences shown as SEQ ID No.1-36, wherein the primer probe group can be used for preparing products for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP. When the invention detects seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP, the invention adopts a fluorescence PCR method, takes the human housekeeping gene GAPDH gene as an internal standard gene as a tube, and the primer probe group as a tube, so that the seven respiratory pathogens can be detected by two tubes within 1 hour, the invention has the advantages of convenient operation, higher sensitivity and stronger specificity, and is not interfered by other microorganisms.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer probe group, application and a kit for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP by multiplex PCR.
Background
Clinically, respiratory tract infections are classified into upper respiratory tract infections and lower respiratory tract infections according to the infection sites. Upper respiratory tract infections are a common acute respiratory tract disease, usually accompanied by acute inflammation of the nasal cavity, pharynx or larynx, most of which are viral infections except for a few bacterial infections. Lower respiratory tract infections include tracheitis, bronchitis, pneumonia and other diseases. Viruses causing respiratory diseases account for over 70%, common viruses are: influenza virus, respiratory syncytial virus, adenovirus, parainfluenza virus, and the like. Because the clinical manifestations of the respiratory tract pathogens after infection are similar, the pathogens are difficult to be clearly caused by the clinical manifestations, the comprehensive, early, accurate and rapid pathogen definition is facilitated, the clinical reasonable medication is facilitated, the targeted treatment is realized, and the accurate diagnosis and treatment of the diseases is realized.
The respiratory tract infection pathogen detection method comprises separation culture, antigen detection, nucleic acid detection, antibody detection and the like, and has respective advantages and disadvantages. Because pathogen isolation and culture are time-consuming and laborious and have low sensitivity, serum antibody detection is not recommended due to the window period, so that the main laboratory diagnostic methods of respiratory tract infection pathogens are antigen detection and nucleic acid detection. The nucleic acid detection method has higher sensitivity and specificity in different disease periods, so that the nucleic acid detection method can be rapidly popularized and applied in clinic and becomes a main method for diagnosing respiratory tract infection pathogens. The reasonable use of multiple nucleic acid detection is not only beneficial to the etiology auxiliary diagnosis of respiratory tract infection, but also provides powerful support for the differential diagnosis of pathogens and the prevention and control of nosocomial infection, and the metagenomic sequencing technology (mNGS) provides possibility for the definition of unknown pathogens.
The current nucleic acid detection method for respiratory diseases mainly comprises the following steps: isothermal amplification techniques, multiplex fluorescent quantitative PCR, PCR capillary electrophoresis, dual amplification (RNA isothermal amplification+multi-biotin signal amplification) techniques, high resolution melting curve analysis (HRM), and the like. The isothermal amplification technology has the advantages of simple operation, high speed, high efficiency, specificity and no need of special equipment, and is more suitable for basic level and field detection. Representative techniques are loop-mediated isothermal amplification (loop mediated iso-thermal amplification, LAMP) and nucleic acid sequence-dependent amplification (nucleic acid sequence based amplification, NASBA). LAMP uses BstDNA polymerase with strand displacement properties to efficiently, rapidly, specifically amplify target sequences under isothermal conditions. Respiratory pathogen detection methods developed based on the LAMP technology have been commercialized. NASBA technology is an isothermal amplification method for detecting RNA, and amplification is generally accomplished at 42 ℃ and is accomplished by reverse transcriptase, RNase H, T RVA polymerase and sequence-specific primers. NASBA is particularly useful for detection of RNA viruses. Currently, there are methods for detecting common respiratory pestilential toxins and haemophilus influenzae, neisseria meningitidis and streptococcus pneumoniae based on NASBA technology for aiding diagnosis. The identification of the amplified product can be detected by color, turbidity colloidal gold technology or by combining with chemiluminescence, fluorescence and chip detection equipment. However, the stability of the amplification system and the maturity of the method by the isothermal amplification technology are inferior to those of the traditional PCR method. The LAMP technology has high requirement on primer design, limits the length of amplified target sequences, and generally cannot amplify long fragments; the NASBA technology has relatively complex reaction system and components, needs multiple enzyme reactions and increases the detection cost.
The nucleic acid amplification technology based on PCR is the most common nucleic acid diagnosis method for detecting the nucleic acid of the respiratory tract pathogen at present because of the advantages of high efficiency, specificity, flexible use, relatively simple operation and low cost. Multiple target design targeting multiple pathogens and research and development applications of integrated equipment for nucleic acid extraction, amplification and result reading highlight the advantages of nucleic acid detection in multiple rapid detection of pathogens. The nucleic acid detection covering common pathogens and drug resistance genes of respiratory tract infection can provide a basis for pathogen diagnosis in clinic, and is helpful for optimizing diagnosis and treatment. According to the technical platform for analyzing PCR products, the method can be divided into a capillary electrophoresis method, a fluorescence method, a mass spectrometry method and the like, wherein the fluorescence method is the most commonly used method.
The PCR capillary electrophoresis method is to analyze PCR products by utilizing capillary electrophoresis, wherein the same PCR reaction system comprises a plurality of pairs of primers (one primer of each target point is provided with a fluorescent mark at the 5' end), each primer is specifically combined with a corresponding target gene, and fragments with different lengths are generated by amplification; the PCR products with different fragment lengths are separated and identified by a capillary electrophoresis system, so that the pathogen is identified. Its advantages are high sensitivity and flux, high repeatability, and high pollution risk.
And (3) carrying out PCR amplification on the DNA to be detected by using a nucleic acid mass spectrometry method, purifying an obtained PCR amplification product by using an astaxanthin enzyme to remove unbound dNTPs, then carrying out single-base extension on the amplification product by using PCR, enriching the extension product by using magnetic beads after extension, detecting the extension product by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, and analyzing data by distinguishing the time of flight. Finally, whether the sequence of the target area is changed is judged through computer software analysis, and then the target area is used for clinical auxiliary diagnosis. The nucleic acid mass spectrometry has multiple, accurate and high flux, and is particularly suitable for molecular diagnosis of complex and multi-target diseases. The disadvantage is that the repeatability is slightly poor and the operating conditions are strictly controlled. And meanwhile, the problems of memory effect, pollution, high price, complex operation and the like generated by the ion source are solved.
The PCR dissolution curve method needs to carry out PCR amplification firstly and then dissolution curve analysis after amplification, and the method has the advantages of long time consumption, small change of the Tm value of a single probe, high-resolution instrument and severe requirement on the instrument.
Chinese patent CN 110964854A discloses a double amplification method. The pathogen nucleic acid fragment is amplified by cleaving the collected sample with a cell lysate and then reverse transcribing and transcribing the pathogen nucleic acid fragment with a reverse transcriptase and a T7RNA polymerase. The amplified RNA product is added into the microwell coated with the coated probe for hybridization, and a specific probe and an amplifying probe corresponding to the coated probe are added. Wherein the coated probe can be complementarily paired with one end sequence of CES series probe of each index, and the other end of CES series probe can be combined with RNA product to anchor the RNA product into the microwell; one end of each LES series probe of each index can be combined with the RNA product, and the other end is combined with the amplifying probe, so that the signal amplifying process is realized. The amplified probe marked with the multi-biotin is combined with a streptavidin-HRP enzyme-linked compound to finally form a coated probe-specific probe-RNA amplified product-specific probe-amplified probe-streptavidin-HRP enzyme-linked compound complex, and finally an HRP enzyme chemiluminescent substrate is added for detection on a chemiluminescent instrument. Detection of pathogen nucleic acid is achieved. The defect is high cost and is not suitable for hospital outpatient screening.
Real-time fluorescent quantitative PCR (real-time PCR) achieves a qualitative to quantitative jump in PCR, using fluorescent reporter molecules to detect the products of each round of PCR amplification.
The method combines the steps of nucleic acid amplification and detection, does not need gel electrophoresis of amplified products, and has the advantages of simplicity, high specificity and sensitivity, good repeatability, accurate quantification, high speed and the like. Compared with the conventional PCR reagent, the Taqman fluorescent probe technology has one more oligonucleotide probe, the probe has one fluorescent luminous group and one fluorescent quenching group, and the complete probe has no fluorescence after being excited by a specific light source. In the PCR process, taq enzyme can degrade a specific fluorescent probe combined with a template through own 5 '-3' exonuclease activity while extending a DNA chain, so that a fluorescent reporter group is separated from a quenching group, and the separated fluorescent reporter group generates fluorescence under the excitation of a specific light source. And (3) carrying out qualitative analysis on the unknown template by monitoring the change of the fluorescence signal in the whole PCR process. Most of the products are developed by the method at present, but detection targets and detection sensitivities of all companies are different, and the problems of inaccurate detection and lower sensitivity still exist.
Disclosure of Invention
Therefore, the invention aims to provide a primer probe group, application and kit for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP by multiplex PCR, which have the advantages of higher sensitivity and stronger specificity in detecting seven respiratory pathogens and are not interfered by other microorganisms.
The invention solves the technical problems by the following technical means:
in a first aspect, the invention provides a primer probe set for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP by multiplex PCR, comprising:
primer Influenza A-F with nucleotide sequence shown as SEQ ID NO.1, primer Influenza A-R with nucleotide sequence shown as SEQ ID NO.2, probe Influenza A-P with nucleotide sequence shown as SEQ ID NO. 3;
primer Influenza B-F with nucleotide sequence shown as SEQ ID NO.4, primer Influenza B-R with nucleotide sequence shown as SEQ ID NO.5, probe Influenza B-P with nucleotide sequence shown as SEQ ID NO. 6;
primers HPIV1-F with nucleotide sequence shown as SEQ ID NO.7, primers HPIV1-R with nucleotide sequence shown as SEQ ID NO.8, and probes HPIV1-P with nucleotide sequence shown as SEQ ID NO. 9;
primer HPIV2-F with nucleotide sequence shown as SEQ ID NO.10, primer HPIV2-R with nucleotide sequence shown as SEQ ID NO.11, probe HPIV2-P with nucleotide sequence shown as SEQ ID NO. 12;
primer HPIV3-F with nucleotide sequence shown as SEQ ID NO.13, primer HPIV3-R with nucleotide sequence shown as SEQ ID NO.14, probe HPIV3-P with nucleotide sequence shown as SEQ ID NO. 15;
primer HPIV4-F with nucleotide sequence shown as SEQ ID NO.16, primer HPIV4-R with nucleotide sequence shown as SEQ ID NO.17, probe HPIV4-P with nucleotide sequence shown as SEQ ID NO. 18;
a primer CP-F with a nucleotide sequence shown as SEQ ID NO.19, a primer CP-R with a nucleotide sequence shown as SEQ ID NO.20, and a probe CP-P with a nucleotide sequence shown as SEQ ID NO. 21;
a primer ADV-be-F with a nucleotide sequence shown as SEQ ID No.22, a primer ADV-be-R with a nucleotide sequence shown as SEQ ID No.23, and a probe ADV-be-P with a nucleotide sequence shown as SEQ ID No. 24;
a primer ADV-c-F with a nucleotide sequence shown as SEQ ID No.25, a primer ADV-c-R with a nucleotide sequence shown as SEQ ID No.26, and a probe ADV-c-P with a nucleotide sequence shown as SEQ ID No. 27;
primer RSVA-F with nucleotide sequence shown as SEQ ID NO.28, primer RSVA-R with nucleotide sequence shown as SEQ ID NO.29, probe RSVA-P with nucleotide sequence shown as SEQ ID NO. 30;
primer RSVB-F with nucleotide sequence shown as SEQ ID NO.31, primer RSVB-R with nucleotide sequence shown as SEQ ID NO.32, probe RSVB-P with nucleotide sequence shown as SEQ ID NO. 33;
primer MP-F with nucleotide sequence shown as SEQ ID NO.34, primer MP-R with nucleotide sequence shown as SEQ ID NO.35, probe MP-P with nucleotide sequence shown as SEQ ID NO. 36.
In a second aspect, the invention provides the use of a primer-probe set as described above in the preparation of a product for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP.
In a third aspect, the invention provides a kit comprising the primer probe set described above.
The primer probe group for detecting seven respiratory pathogens of the IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP by the multiplex PCR and the kit containing the primer probe group are carried out by adopting a fluorescence PCR method when the seven respiratory pathogens of the IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP are detected, the housekeeping gene GAPDH is taken as an internal standard gene as a tube, the primer probe group is taken as a tube, and the seven respiratory pathogens can be detected by two tubes within 1 hour, so that the operation is convenient.
The detection sensitivity of the kit of the primer probe set designed by the invention can reach 500 copies/mL, and the kit has the advantages of high sensitivity and strong specificity, is not interfered by other microorganisms, and is compared by NCBI blast, and pathogenic microorganisms such as: the binding of the primer and the probe to the BoKavirus, cytomegalovirus, herpes simplex virus type I, varicella zoster virus, EB virus, legionella pneumophila, escherichia coli, staphylococcus aureus, candida albicans and the like has not been predicted, and a correlation curve has not been detected in the subsequent pathogen fluorescence quantitative PCR detection, which shows that the specificity of the primer and the probe is good.
Drawings
FIG. 1 is a PCR amplification diagram for detecting infection with influenza A virus H1N1 (novel influenza A H1N1 virus (2009), seasonal H1N1 influenza virus); FIG. 2 is a diagram of PCR amplification for detecting H3N2 infection; FIG. 3 is a PCR amplification diagram for detecting H5N1 infection; FIG. 4 is a PCR amplification plot for detecting H7N9 infection; FIG. 5 is a PCR amplification plot for detection of influenza B infection by Yamagata; FIG. 6 is a PCR amplification plot for detecting Victoria infection; FIG. 7 is a PCR amplification diagram for detecting human parainfluenza virus type 1 infection; FIG. 8 is a PCR amplification diagram for detecting human parainfluenza virus type 2 infection; FIG. 9 is a PCR amplification diagram for detecting human parainfluenza virus type 3 infection; FIG. 10 is a PCR amplification diagram for detecting human parainfluenza virus type 4 infection; FIG. 11 is a PCR amplification plot for detection of Chlamydia pneumoniae infection; FIG. 12 is a PCR amplification plot for detection of adenovirus B infection; FIG. 13 is a PCR amplification plot for detection of adenovirus C infection; FIG. 14 is a PCR amplification plot for detection of adenovirus E infection; FIG. 15 is a PCR amplification plot for detection of respiratory syncytial virus A infection; FIG. 16 is a PCR amplification plot for detection of respiratory syncytial virus B infection; FIG. 17 is a PCR amplification plot for detection of mycoplasma pneumoniae infection; FIG. 18 is a PCR amplification plot of the lowest detection limit (500 copies/mL) of influenza A virus H1N 1; FIG. 19 is a PCR amplification plot of the lowest detection limit (500 copies/mL) of influenza A virus H3N 2; FIG. 20 is a PCR amplification plot of the lowest detection limit (500 copies/mL) of influenza A virus H5N 1; FIG. 21 is a PCR amplification plot of the lowest detection limit (500 copies/mL) of influenza A virus H7N 9; FIG. 22 is a PCR amplification plot of the minimum detection limit (500 copies/mL) of influenza B virus Yamagata; FIG. 23 is a PCR amplification plot of the minimum detection limit (500 copies/mL) of influenza B virus Victoria; FIG. 24 is a PCR amplification plot of human parainfluenza virus type 1 minimum detection limit (500 copies/mL); FIG. 25 is a PCR amplification plot of human parainfluenza virus type 2 minimum detection limit (500 copies/mL); FIG. 26 is a PCR amplification plot of human parainfluenza virus type 3 minimum detection limit (500 copies/mL); FIG. 27 is a PCR amplification plot of human parainfluenza virus type 4 minimum detection limit (500 copies/mL); FIG. 28 is a PCR amplification plot showing the minimum detection limit (500 copies/mL) of Chlamydia pneumoniae; FIG. 29 is a PCR amplification plot of adenovirus B minimum detection limit (500 copies/mL); FIG. 30 is a PCR amplification plot of adenovirus C minimum detection limit (500 copies/mL); FIG. 31 is a PCR amplification plot of adenovirus E minimum detection limit (500 copies/mL); FIG. 32 is a PCR amplification plot of respiratory syncytial virus A minimum detection limit (500 copies/mL); FIG. 33 is a PCR amplification plot of respiratory syncytial virus B minimum detection limit (500 copies/mL); FIG. 34 is a PCR amplification plot showing the minimum detection limit (500 copies/mL) of Mycoplasma pneumoniae.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The following examples were conducted under conventional conditions or conditions recommended by the manufacturer, without specifying the specific conditions. The raw materials, equipment or instruments used are conventional products commercially available without identifying the manufacturer.
Firstly, searching genome information of 7 respiratory pathogens issued by NCBI, searching different genome sequences of each virus, and then using DNAMAN software to perform sequence comparison on the genome sequences; searching out genes with genome specificity, and comparing by using blast software to determine sequences which have no homology specificity between the specific genes in the genome and NCBI library as candidate genes; searching candidate genes in target virus genome data, determining conservation of upstream and downstream genes, avoiding high mutation areas, and marking variable bases in the form of degenerate bases; and verifying the candidate genes, and taking the candidate sequences with specificity and conservation as target genes. And (3) after designing multiple primers and probes on the target gene position, optimizing a multiple PCR reaction system and conditions to obtain the multiple PCR reaction system and conditions.
The problem of co-amplification of primer probes of different target genes in one reaction system is fully considered in the design process of the primer probe set, so that the Tm value consistency and GC content uniformity are comprehensively considered in the design of the primer probe, and meanwhile, the conditions of hairpin structures, primer dimers and the like are avoided as far as possible, so that the probability of simultaneous amplification of different primer probes in the later stage is ensured. Finally, a set of primer probe sequences provided by the invention is obtained, comprising:
primer Influenza A-F with nucleotide sequence shown as SEQ ID NO.1, primer Influenza A-R with nucleotide sequence shown as SEQ ID NO.2, probe Influenza A-P with nucleotide sequence shown as SEQ ID NO. 3;
primer Influenza B-F with nucleotide sequence shown as SEQ ID NO.4, primer Influenza B-R with nucleotide sequence shown as SEQ ID NO.5, probe Influenza B-P with nucleotide sequence shown as SEQ ID NO. 6;
primers HPIV1-F with nucleotide sequence shown as SEQ ID NO.7, primers HPIV1-R with nucleotide sequence shown as SEQ ID NO.8, and probes HPIV1-P with nucleotide sequence shown as SEQ ID NO. 9;
primer HPIV2-F with nucleotide sequence shown as SEQ ID NO.10, primer HPIV2-R with nucleotide sequence shown as SEQ ID NO.11, probe HPIV2-P with nucleotide sequence shown as SEQ ID NO. 12;
primer HPIV3-F with nucleotide sequence shown as SEQ ID NO.13, primer HPIV3-R with nucleotide sequence shown as SEQ ID NO.14, probe HPIV3-P with nucleotide sequence shown as SEQ ID NO. 15;
primer HPIV4-F with nucleotide sequence shown as SEQ ID NO.16, primer HPIV4-R with nucleotide sequence shown as SEQ ID NO.17, probe HPIV4-P with nucleotide sequence shown as SEQ ID NO. 18;
a primer CP-F with a nucleotide sequence shown as SEQ ID NO.19, a primer CP-R with a nucleotide sequence shown as SEQ ID NO.20, and a probe CP-P with a nucleotide sequence shown as SEQ ID NO. 21;
a primer ADV-be-F with a nucleotide sequence shown as SEQ ID No.22, a primer ADV-be-R with a nucleotide sequence shown as SEQ ID No.23, and a probe ADV-be-P with a nucleotide sequence shown as SEQ ID No. 24;
a primer ADV-c-F with a nucleotide sequence shown as SEQ ID No.25, a primer ADV-c-R with a nucleotide sequence shown as SEQ ID No.26, and a probe ADV-c-P with a nucleotide sequence shown as SEQ ID No. 27;
primer RSVA-F with nucleotide sequence shown as SEQ ID NO.28, primer RSVA-R with nucleotide sequence shown as SEQ ID NO.29, probe RSVA-P with nucleotide sequence shown as SEQ ID NO. 30;
primer RSVB-F with nucleotide sequence shown as SEQ ID NO.31, primer RSVB-R with nucleotide sequence shown as SEQ ID NO.32, probe RSVB-P with nucleotide sequence shown as SEQ ID NO. 33;
primer MP-F with nucleotide sequence shown as SEQ ID NO.34, primer MP-R with nucleotide sequence shown as SEQ ID NO.35, probe MP-P with nucleotide sequence shown as SEQ ID NO. 36.
In order to ensure the accuracy of template extraction in the test and the concentration of the preliminary estimated template, the normal operation of the whole reaction system is monitored, and a housekeeping gene is set as an internal control of the PCR detection system. One of GAPDH (glycal dehyde-3-phosphate-dehydrogenase), ACTB (beta-actin), beta-globin and the like genes can be selected. In the present invention, GAPDH gene is selected as an internal reference gene.
As the fluorescent modification group for the probe, one or more of FAM (6-carboxyfluorescein), HEX (hexachloro-6-methylfluorescein), VIC, ROX, cy (TY TM 563) and Cy5 (TY TM 665) can be used, and each modification can be exchanged.
Example 1
Based on the above, first, screening of primer probes is performed. The detection primers and probe designs for the seven respiratory pathogens are shown in table 1.
Table 1:
the primer and probe designs for the human housekeeping gene are shown in Table 2.
Table 2:
secondly, a kit is preliminarily designed, wherein the kit comprises a primer, a probe and a PCR reagent component, the PCR reagent component comprises dNTP, DNA polymerase, reverse transcriptase, UNG enzyme and PCR buffer solution, and the following operations are carried out:
(1) Screening the primers and the probes based on the kit with the same component concentration and the same PCR reaction condition, observing the curve, the fluorescence value and the ct value of fluorescence quantitative PCR, and selecting the optimal primers and probes from the curve, the fluorescence value and the ct value;
(2) Screening dNTP, DNA polymerase, reverse transcriptase, UNG enzyme and PCR buffer solution in the kit based on the most selected primer and probe, comparing the enzymes of different manufacturers and different types, observing the curve, fluorescence value and ct value of fluorescence quantitative PCR, and selecting the optimal enzyme type;
(3) Screening the concentration and proportion of the primer and the probe, observing the curve, the fluorescence value and the ct value of fluorescence quantitative PCR, and selecting the optimal concentration and proportion of the primer and the probe;
the amount of the reaction system was 20. Mu.L (15. Mu.L of the reaction solution, 5. Mu.L of the template was added). Based on the above, the preparation method of the kit for detecting seven respiratory pathogens of the invention comprises the following steps:
(1) And (3) delivering the screened primer and probe sequences to a gene synthesis company for synthesis to obtain dry powder.
(2) The primer and probe sequences were diluted to 50. Mu.M with the recommended addition of 1 XTE.
(3) Other fluorescent quantitative PCR reagent components such as dntps, DNA polymerase, reverse transcriptase, UNG enzyme, PCR buffer, etc. were purchased.
(4) Mixing the primer probe and each component according to the concentration of the reaction system after screening, preparing a reaction solution, and sub-packaging and freezing.
Finally, a reaction program screening was performed, and in the kit for detecting seven respiratory pathogens of the present invention, the fluorescent quantitative PCR reaction conditions are shown in Table 3.
Table 3:
after optimization, the fluorescent quantitative PCR reaction conditions are shown in Table 4.
Table 4:
the seven respiratory pathogen detection sequences of the screened multiplex PCR detection IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP are shown in Table 5:
table 5:
the detection sequences of the screened GAPDH genes are shown in table 6.
Table 6:
| GAPDH-F | 185 | GCACCGTCAAGGCTGAGAAC | SEQ ID NO.37 |
| GAPDH-R | 188 | CAAAGCACATTTCTTCCATTCTGT | SEQ ID NO.38 |
| GAPDH-P | 191 | CY5-AAATCCCATCACCATCTTCCAGGAGTGAG-BHQ1 | SEQ ID NO.39 |
example 2
This example provides a kit for detecting 7 respiratory pathogen infections and partial typing comprising the primers and probes screened in example 1, the kit having the reagent formulations shown in Table 7.
Table 7:
according to the concentration formula, adding various reaction liquid components, uniformly mixing, and then sub-packaging and preserving. Wherein GAPDH-F, GAPDH-R, GAPDH-P is a detection primer and a probe of a human housekeeping gene GAPDH. Influenza A-F, influenza A-R, influenza A-P, influenza B-F, influenza B-R, influenza B-P, HPIV1-F, HPIV1-R, HPIV1-R, HPIV2-F, HPIV2-R, HPIV-P, HPIV3-F, HPIV-R, HPIV3-P, HPIV-F, HPIV-R, HPIV4-P, CP-F, CP-R, CP-P, ADV-be-F, ADV-be-R, ADV-be-P, ADV-c-F, ADV-c-R, ADV-c-P, RSVA-F, RSVA-R, RSVA-P, RSVB-F, RSVB-R, RSVB-P, MP-F, MP-R, MP-P are detection primers and probes for detecting whether seven respiratory pathogens are infected or not, and different pathogens are distinguished through different fluorescent signals.
Example 3
The present example provides a method of using the kit of example 2, mixing the primer probe composition therein with the template DNA/RNA of the sample to be detected, and performing RT-qPCR detection. The GAPDH primer, the probe and the target region on the template DNA are combined, and a Ct value is obtained during detection to judge whether the whole reaction system works normally or not; the remaining primers bind to the region where the probe and template DNA are located and, upon detection, a Ct value is obtained to determine the presence or absence of the seven respiratory pathogen infections.
The application method of the kit specifically comprises the following steps:
(1) Extracting nucleic acid of a sample to be detected.
(2) And (3) preparation of a reagent: preparation of PCR amplification reaction solution was carried out in accordance with 15. Mu.L× (number of samples to be detected+positive control+negative control) number of samples to be detected. The samples were equally distributed into 8 rows of tubes in a volume of 15. Mu.L per tube.
(3) 5 mu L of PCR amplified template obtained by extracting nucleic acid is added into the reagent, positive control and negative control are amplified simultaneously, and the amplification is carried out after short centrifugation to an amplification region, and the amplification procedure is shown in Table 8.
Table 8:
(4) Interpretation of the results:
referring to fig. 1 to 17, the method for determining whether to infect seven respiratory pathogens by Ct value difference is as follows:
the internal parameters (CY 5 signals) of the sample B to be detected all have obvious amplified signals and Ct CY5 <40, the subsequent interpretation can be made:
if the B tube internal reference CY5 channel has no obvious amplified signal or Ct CY5 >40, the sample is not effectively extracted or sampling is not standard, and the result is invalid and the detection needs to be resampled.
The following result interpretation criteria are established in that the internal reference has a significant amplified signal and Ct CY5 <40, if FAM in the A tube has obvious amplified signal or Ct FAM <40, indicating that the sample is infected with influenza a virus; if there is a significant amplification signal or Ct in ROX in the A tube FAM <40, indicating that the sample has influenza b virus infection; if the VIC in the A tube has obvious amplified signal or Ct FAM <40, indicating that the sample has infection by human parainfluenza virus; if CY5 in A tube has obvious amplified signal or Ct FAM <40, indicating that the sample has an infection with Chlamydia pneumoniae; if FAM in B tube has obvious amplified signal or Ct FAM <40, indicating that the sample has adenovirus B/C/E infection; if there is a significant amplification signal or Ct in the ROX in the B tube FAM <40, indicating that the sample has respiratory syncytial virus infection; if the VIC in the B tube has obvious amplified signal or Ct FAM <40, the samples were then indicated for infection with mycoplasma pneumoniae. In addition to the internal reference channel, if there is a significant amplification signal or Ct in both the A and B tubes FAM <40, it indicates that the sample has more than 2 mixed infections.
Example 4
The sensitivity test was performed in this example, i.e., the following tests were performed using the kit of example 2:
(1) By synthesizing the plasmid of the influenza A virus H1N1, carrying out qPCR amplification on samples with plasmid concentration diluted to different concentration gradients, and the experimental result is shown in figure 18, and the obvious amplification curve appears as can be seen from figure 18, which shows that the detection sensitivity of the influenza A virus H1N1 primer probe set designed by the invention can reach 500 copies/mL.
(2) By synthesizing the plasmid of the influenza A virus H3N2, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 19, and the obvious amplification curve appears in figure 19, which shows that the detection sensitivity of the influenza A virus H3N2 primer probe set designed by the invention can reach 500 copies/mL.
(3) By synthesizing the plasmid of the influenza A virus H5N1, carrying out qPCR amplification on samples with plasmid concentration diluted to different concentration gradients, and the experimental result is shown in figure 20, and the obvious amplification curve appears in figure 20, which shows that the detection sensitivity of the influenza A virus H5N1 primer probe set designed by the invention can reach 500 copies/mL.
(4) By synthesizing the plasmid of the influenza A virus H7N9, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 21, and the obvious amplification curve appears as can be seen from figure 21, which shows that the detection sensitivity of the influenza A virus H7N9 primer probe set designed by the invention can reach 500 copies/mL.
(5) By synthesizing the plasmid of the influenza B virus Yamagata, carrying out qPCR amplification on samples with plasmid concentration diluted to different concentration gradients, and the experimental result is shown in figure 22, and the obvious amplification curve appears in figure 22, which shows that the detection sensitivity of the primer probe set of the influenza B virus Yamagata designed by the invention can reach 500 copies/mL.
(6) By synthesizing the plasmid of the influenza B virus Victoria, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 23, and the obvious amplification curve appears as can be seen from figure 23, which shows that the detection sensitivity of the influenza B virus Victoria primer probe set designed by the invention can reach 500 copies/mL.
(7) By synthesizing the human parainfluenza virus type 1 plasmid, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 24, and the obvious amplification curve appears in figure 24, which shows that the detection sensitivity of the human parainfluenza virus type 1 primer probe set designed by the invention can reach 500 copies/mL.
(8) By synthesizing the human parainfluenza virus type 2 plasmid, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 25, and the obvious amplification curve appears in figure 25, which shows that the detection sensitivity of the human parainfluenza virus type 2 primer probe set designed by the invention can reach 500 copies/mL.
(9) By synthesizing the human parainfluenza virus 3 type plasmid, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 26, and the obvious amplification curve appears in figure 26, which shows that the detection sensitivity of the human parainfluenza virus 3 type primer probe set designed by the invention can reach 500 copies/mL.
(10) By synthesizing the human parainfluenza virus type 4 plasmid, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 27, and the obvious amplification curve appears in figure 27, which shows that the detection sensitivity of the human parainfluenza virus type 4 primer probe set designed by the invention can reach 500 copies/mL.
(11) The plasmid of the Chlamydia pneumoniae is synthesized, the plasmid concentration is diluted into samples with different concentration gradients for qPCR amplification, the experimental result is shown in figure 28, and the obvious amplification curve appears in figure 28, which shows that the detection sensitivity of the designed Chlamydia pneumoniae primer probe set can reach 500 copies/mL.
(12) By synthesizing the plasmid of the adenovirus B, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 29, and the obvious amplification curve appears in figure 29, which shows that the detection sensitivity of the adenovirus B primer probe set designed by the invention can reach 500 copies/mL.
(13) By synthesizing the plasmid of the adenovirus C, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 30, and the obvious amplification curve appears in figure 30, which shows that the detection sensitivity of the adenovirus C primer probe set designed by the invention can reach 500 copies/mL.
(14) By synthesizing adenovirus E plasmid, qPCR amplification is carried out on samples with plasmid concentration diluted to different concentration gradients, the experimental result is shown in figure 31, and the obvious amplification curve appears in figure 31, which shows that the detection sensitivity of the adenovirus E primer probe set designed by the invention can reach 500 copies/mL.
(15) The plasmid concentration is diluted into samples with different concentration gradients for qPCR amplification through the plasmid of the respiratory syncytial virus A, the experimental result is shown in figure 32, and the obvious amplification curve appears in the figure 32, which shows that the detection sensitivity of the respiratory syncytial virus A primer probe set designed by the invention can reach 500 copies/mL.
(16) The plasmid concentration is diluted into samples with different concentration gradients for qPCR amplification through the plasmid of the respiratory syncytial virus B, the experimental result is shown in figure 33, and the obvious amplification curve appears as can be seen from figure 33, which shows that the detection sensitivity of the respiratory syncytial virus B primer probe set designed by the invention can reach 500 copies/mL.
(17) The plasmid concentration of the mycoplasma pneumoniae is diluted into samples with different concentration gradients to carry out qPCR amplification, the experimental result is shown in figure 34, and the obvious amplification curve appears in figure 34, which shows that the detection sensitivity of the mycoplasma pneumoniae primer probe set designed by the invention can reach 500 copies/mL.
The test shows that the detection sensitivity of the kit of the primer probe set designed by the invention can reach 500 copies/mL, and the kit has the advantages of high sensitivity and strong specificity. By NCBI blast alignment, pathogenic microorganisms that interfere in cross-talk such as: the binding of the primer and the probe to the BoKavirus, cytomegalovirus, herpes simplex virus type I, varicella zoster virus, EB virus, legionella pneumophila, escherichia coli, staphylococcus aureus, candida albicans and the like has not been predicted, and a correlation curve has not been detected in the subsequent pathogen fluorescence quantitative PCR detection, which shows that the specificity of the primer and the probe is good.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention. The technology, shape, and construction parts of the present invention, which are not described in detail, are known in the art.
Claims (9)
1. A primer probe set for multiplex PCR detection of seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP, comprising:
primer Influenza A-F with nucleotide sequence shown as SEQ ID NO.1, primer Influenza A-R with nucleotide sequence shown as SEQ ID NO.2, probe Influenza A-P with nucleotide sequence shown as SEQ ID NO. 3;
primer Influenza B-F with nucleotide sequence shown as SEQ ID NO.4, primer Influenza B-R with nucleotide sequence shown as SEQ ID NO.5, probe Influenza B-P with nucleotide sequence shown as SEQ ID NO. 6;
primers HPIV1-F with nucleotide sequence shown as SEQ ID NO.7, primers HPIV1-R with nucleotide sequence shown as SEQ ID NO.8, and probes HPIV1-P with nucleotide sequence shown as SEQ ID NO. 9;
primer HPIV2-F with nucleotide sequence shown as SEQ ID NO.10, primer HPIV2-R with nucleotide sequence shown as SEQ ID NO.11, probe HPIV2-P with nucleotide sequence shown as SEQ ID NO. 12;
primer HPIV3-F with nucleotide sequence shown as SEQ ID NO.13, primer HPIV3-R with nucleotide sequence shown as SEQ ID NO.14, probe HPIV3-P with nucleotide sequence shown as SEQ ID NO. 15;
primer HPIV4-F with nucleotide sequence shown as SEQ ID NO.16, primer HPIV4-R with nucleotide sequence shown as SEQ ID NO.17, probe HPIV4-P with nucleotide sequence shown as SEQ ID NO. 18;
a primer CP-F with a nucleotide sequence shown as SEQ ID NO.19, a primer CP-R with a nucleotide sequence shown as SEQ ID NO.20, and a probe CP-P with a nucleotide sequence shown as SEQ ID NO. 21;
a primer ADV-be-F with a nucleotide sequence shown as SEQ ID No.22, a primer ADV-be-R with a nucleotide sequence shown as SEQ ID No.23, and a probe ADV-be-P with a nucleotide sequence shown as SEQ ID No. 24;
a primer ADV-c-F with a nucleotide sequence shown as SEQ ID No.25, a primer ADV-c-R with a nucleotide sequence shown as SEQ ID No.26, and a probe ADV-c-P with a nucleotide sequence shown as SEQ ID No. 27;
primer RSVA-F with nucleotide sequence shown as SEQ ID NO.28, primer RSVA-R with nucleotide sequence shown as SEQ ID NO.29, probe RSVA-P with nucleotide sequence shown as SEQ ID NO. 30;
primer RSVB-F with nucleotide sequence shown as SEQ ID NO.31, primer RSVB-R with nucleotide sequence shown as SEQ ID NO.32, probe RSVB-P with nucleotide sequence shown as SEQ ID NO. 33;
primer MP-F with nucleotide sequence shown as SEQ ID NO.34, primer MP-R with nucleotide sequence shown as SEQ ID NO.35, probe MP-P with nucleotide sequence shown as SEQ ID NO. 36.
2. The primer probe set for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP by multiplex PCR according to claim 1, wherein the 5' end of the probe Influenza a-P is labeled with FAM chromophore;
the 5' end of the probe Influenza B-P is marked with a ROX luminous group;
the 5' end of the probe HPIV1-P is marked with a VIC luminous group;
the 5' end of the probe HPIV2-R is marked with a VIC luminous group;
the 5' end of the probe HPIV3-R is marked with a VIC luminous group;
the 5' end of the probe HPIV4-R is marked with a VIC luminous group;
the 5' end of the probe CP-P is marked with a CY5 luminous group;
the 5' end of the probe ADV-be-P is marked with a FAM luminous group;
the 5' end of the probe ADV-c-P is marked with a FAM luminous group;
the 5' end of the probe RSVA-P is marked with a ROX luminous group;
the 5' end of the probe RSVB-P is marked with a ROX luminous group;
the 5' end of the probe MP-P is marked with a VIC luminous group.
3. Use of a primer probe set according to claim 1 or 2 in the preparation of a product for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP.
4. The use according to claim 3, wherein the product is a detection reagent, a detection kit or a detection chip.
5. A kit comprising the primer probe set according to claim 1 or 2.
6. The kit according to claim 5, further comprising an internal standard gene primer probe composition, wherein the internal standard gene is a human housekeeping gene GAPDH gene.
7. The kit of claim 6, wherein the internal standard gene primer probe composition comprises:
a primer GAPDH-F of the nucleotide sequence shown in SEQ ID NO. 37;
a primer GAPDH-R of the nucleotide sequence shown in SEQ ID NO. 38;
a probe GAPDH-P having the nucleotide sequence shown in SEQ ID NO. 39;
the 5 'end of the probe GAPDH-P is marked with a CY5 luminous group, and the 3' end is marked with a quenching group BHQ1.
8. A kit according to claim 7, wherein the concentration of the primer GAPDH-F, GAPDH-R in the kit is 0.1 μm and the concentration of the probe GAPDH-P is 0.05 μm;
the concentrations of the primers Influenza A-F, influenza A-R, HPIV1-F, HPIV1-R, HPIV2-F, HPIV2-R, HPIV3-F, HPIV-R, HPIV4-F, HPIV4-R, ADV-be-F, ADV-be-R, ADV-c-F, ADV-c-R, RSVA-F, RSVA-R, RSVB-F, RSVB-R and the concentrations of the probes Influenza A-P, HPIV1-P, HPIV2-P, HPIV3-P, HPIV-P, ADV-be-P, ADV-c-P, RSVA-P, RSVB-P are all 0.2 mu M;
the concentration of the primer MP-F, MP-R, CP-F, CP-R is 0.6 mu M, and the concentration of the probe MP-P, CP-P is 0.3 mu M;
the concentrations of the primer Influenza B-F and the primer Influenza B-R are 0.3 mu M, and the concentration of the probe Influenza B-P is 0.15 mu M.
9. The kit according to claim 8, further comprising a PCR reagent component comprising dNTPs, a DNA polymerase, a UNG enzyme, a reverse transcriptase, and a PCR buffer, wherein the concentration of the DNA polymerase is 0.2 to 0.4U, the concentration of the reverse transcriptase is 0.2 to 0.4U, the concentration of the UNG enzyme is 0.1 to 0.2U, and the concentration of the dNTPs is 2.0 to 2.5. Mu.M.
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| CN202311262068.2A CN117568524A (en) | 2023-09-27 | 2023-09-27 | Primer probe group for detecting seven respiratory pathogens of IFV-A, IFV-B, RSV, HPIV, HAdV, MP, CP by multiplex PCR, application and kit |
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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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| Publication number | Priority date | Publication date | Assignee | Title |
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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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