CN110408726B - Method for detecting 29 respiratory pathogens by using Taqman low-density microfluidic chip technology - Google Patents
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Abstract
The invention discloses a method for detecting 29 respiratory pathogens by using a Taqman low-density microfluidic chip technology. The Taqman low-density microfluidic chip (TAC) detection technology for 29 respiratory pathogens, which is established by the invention, fully utilizes the advantages of MGB probe detection, overcomes the defect of multiple detection of the traditional respiratory pathogens, can realize multi-target detection of the 29 respiratory pathogens on one sample within two hours, and has good sensitivity and specificity. The invention provides a rapid, accurate and high-flux technical means for monitoring and outbreak investigation of respiratory diseases in China.
Description
Technical Field
The invention relates to a method for detecting 29 respiratory pathogens by using a Taqman low-density microfluidic chip technology.
Background
Respiratory diseases include acute respiratory infections and community-acquired pneumonia, which pose serious threats and challenges to human health. Among them, respiratory pathogens closely related to the occurrence of two diseases include viruses, bacteria, mycoplasma, chlamydia and rickettsia, etc., and many pathogens cause similar clinical symptoms, so misdiagnosis easily occurs in pathogen diagnosis, and the phenomenon of antibiotic abuse is ubiquitous. Timely and accurate differential diagnosis of pathogens is beneficial to prevention and treatment of respiratory diseases, and the burden of social medical health is relieved.
The traditional respiratory pathogen detection technology mainly comprises detection technical means such as bacteria and virus culture, antigen-antibody reaction, immunofluorescence and common PCR. However, these above techniques have certain drawbacks, more or less: the culture period of bacteria or cells is long and the requirement is high, while pathogen diagnosis needs certain timeliness, and the bacteria or cell culture cannot meet the requirement of short-time rapid detection; the antigen-antibody reaction sensitivity and specificity are low, false positive is easy to occur, and the method is not suitable for detecting mixed infection of various pathogens; the common gel electrophoresis has low sensitivity, sometimes has fuzzy bands, is not accurate enough in judgment, and is very easy to cause misdiagnosis.
The Real-time PCR technology has the advantages of high sensitivity and specificity, short detection time and accuracy, and becomes a main technical method for detecting pathogens at home and abroad. In addition, the kit is mainly used for detecting single respiratory tract pathogen infection, and a few methods for simultaneously detecting different respiratory tract pathogens are used, so that the kit is not beneficial to simultaneously screening multiple pathogen infections. In the multiplex PCR detection, the primer probes for detecting different pathogens are in one reaction tube, so that false positive is easily caused. In addition, in multiplex PCR assays, the alteration of the primer probe to detect one of the pathogens results in an alteration of the overall assay system, requiring a new optimization of the assay system, which makes the assay more complex. In order to change the defects of the above research and improve the capability of rapidly screening various respiratory pathogens, it is necessary to establish a rapid, sensitive and accurate technical system with the capability of rapidly screening various pathogens on site and in a laboratory.
The Taqman low-density microfluidic chip (TAC) technology is an emerging multi-pathogen screening technology, and in a 384-hole microfluidic chip, one sample can realize simultaneous detection of up to 48 targets, so that the Taqman low-density microfluidic chip is favorable for multi-target screening of trace samples.
Disclosure of Invention
The invention aims to provide a method for detecting 29 respiratory pathogens by using a Taqman low-density microfluidic chip technology.
The invention firstly provides a set of primer probes, which comprises 29 sets of primer probe combinations;
the primer probe combination 1 consists of primers shown in sequences 1 and 2 of a sequence table and a probe shown in a sequence 3;
the primer probe combination 2 consists of primers shown in sequences 4 and 5 of a sequence table and a probe shown in a sequence 6;
the primer probe combination 3 consists of primers shown in sequences 7 and 8 of a sequence table and a probe shown in a sequence 9;
the primer probe combination 4 consists of primers shown in sequences 10 and 11 of the sequence table and a probe shown in a sequence 12;
the primer probe combination 5 consists of primers shown in sequences 13 and 14 of a sequence table and a probe shown in a sequence 15;
the primer probe combination 6 consists of primers shown in sequences 16 and 17 of the sequence table and a probe shown in a sequence 18;
the primer probe combination 7 consists of primers shown in sequences 19 and 20 of a sequence table and a probe shown in a sequence 21;
the primer probe combination 8 consists of primers shown in sequences 22 and 23 of the sequence table and a probe shown in a sequence 24;
the primer probe combination 9 consists of primers shown in sequences 25 and 26 of the sequence table and a probe shown in a sequence 27;
the primer probe combination 10 consists of primers shown in sequences 28 and 29 of a sequence table and a probe shown in a sequence 30;
the primer probe combination 11 consists of primers shown in sequences 31 and 32 of a sequence table and a probe shown in a sequence 33;
the primer probe combination 12 consists of primers shown in sequences 34 and 35 of a sequence table and a probe shown in a sequence 36;
the primer probe combination 13 consists of primers shown in sequences 37 and 38 of the sequence table and a probe shown in a sequence 39;
the primer probe combination 14 consists of primers shown in sequences 40 and 41 of a sequence table and a probe shown in a sequence 42;
the primer probe combination 15 consists of primers shown in sequences 43 and 44 of a sequence table and a probe shown in a sequence 45;
the primer probe combination 16 consists of primers shown in sequences 46 and 47 of the sequence table and a probe shown in a sequence 48;
the primer probe combination 17 consists of primers shown in sequences 49 and 50 of the sequence table and a probe shown in a sequence 51;
the primer probe combination 18 consists of primers shown in sequences 52 and 53 of a sequence table and a probe shown in a sequence 54;
the primer probe combination 19 consists of primers shown in sequences 55 and 56 of the sequence table and a probe shown in a sequence 57;
the primer probe combination 20 consists of primers shown in sequences 58 and 59 of a sequence table and a probe shown in a sequence 60;
the primer probe combination 21 consists of primers shown in sequences 61 and 62 of a sequence table and a probe shown in a sequence 63;
the primer probe combination 22 consists of primers shown in sequences 64 and 65 of a sequence table and a probe shown in a sequence 66;
the primer probe combination 23 consists of primers shown in sequences 67 and 68 of the sequence table and a probe shown in a sequence 69;
the primer probe combination 24 consists of primers shown in sequences 70 and 71 of a sequence table and a probe shown in a sequence 72;
the primer probe combination 25 consists of primers shown in sequences 73 and 74 of the sequence table and a probe shown in a sequence 75;
the primer probe combination 26 consists of primers shown in sequences 76 and 77 of the sequence table and a probe shown in a sequence 78;
the primer probe combination 27 consists of primers shown in sequences 79 and 80 of the sequence table and a probe shown in a sequence 81;
the primer probe combination 28 consists of primers shown in sequences 82 and 83 of the sequence table and a probe shown in a sequence 84;
the primer probe combination 29 consists of primers shown in sequences 85 and 86 of the sequence table and a probe shown in a sequence 87.
The set of primer probes further comprises a primer probe combination 30;
the primer probe combination 30 consists of primers shown in sequences 88 and 89 of the sequence table and a probe shown in a sequence 90.
The set of primer probes may further comprise a primer probe combination for detecting IPCO.
Any one of the probes described above is an MGB probe; one end of the probe is marked by a fluorescent group, and the other end of the probe is marked by an MGB group. The probe can be specifically marked by FAM fluorescent group at the 5 'end and MGB group at the 3' end.
In each primer probe combination, the molar ratio of the two primers to the probe is 1.
The use of the primer probe set described above is any one of the following (a 1) to (a 6):
(a1) Identifying 29 respiratory pathogens;
(a2) Preparing products for identifying and identifying 29 respiratory pathogens;
(a3) Identifying whether the pathogen to be detected is one of 29 respiratory tract pathogens;
(a4) Preparing a product for identifying whether the pathogen to be detected is one of 29 respiratory pathogens;
(a5) Identifying whether the sample to be tested is infected with one or more of 29 respiratory pathogens;
(a6) Products were prepared for identifying whether a test sample was infected with one or more of the 29 respiratory pathogens.
The invention also protects the application of the primer probe set as described above, wherein the primer probe set comprises any one of the following (a 1) to (a 6):
(a1) Identifying 29 respiratory pathogens;
(a2) Preparing products for identifying and identifying 29 respiratory pathogens;
(a3) Identifying whether the pathogen to be detected is one of 29 respiratory tract pathogens;
(a4) Preparing a product for identifying whether the pathogen to be detected is one of 29 respiratory pathogens;
(a5) Identifying whether the sample to be tested is infected with one or more of 29 respiratory pathogens;
(a6) Products were prepared for identifying whether a test sample was infected with one or more of the 29 respiratory pathogens.
The invention also protects a kit containing any one of the primer probe sets; the application of the kit is (b 1), (b 2) or (b 3):
(b1) Identifying 29 respiratory pathogens;
(b2) Identifying whether the pathogen to be detected is one of 29 respiratory tract pathogens;
(b3) And (3) identifying whether the sample to be tested is infected with one or more of 29 respiratory pathogens.
The kit also comprises other reagents for detecting 29 respiratory pathogens by using quantitative PCR, and specifically can be reagents in a real-time quantitative One-Step reverse transcription kit, such as 2 xQuant One Step Probe qRT-PCR master Mix, hotMaster Taq polymerase and QuantRTase.
The invention also provides a preparation method of the kit, which comprises the step of packaging each primer probe independently.
The invention also protects a Taqman low-density microfluidic chip; any one of the primer probes set is fixed on the chip.
The Taqman low-density microfluidic chip is used for any one of the following (a 1) to (a 6):
(a1) Identifying 29 respiratory pathogens;
(a2) Preparing products for identifying and identifying 29 respiratory pathogens;
(a3) Identifying whether the pathogen to be detected is one of 29 respiratory tract pathogens;
(a4) Preparing a product for identifying whether the pathogen to be detected is one of 29 respiratory pathogens;
(a5) Identifying whether the sample to be tested is infected with one or more of 29 respiratory pathogens;
(a6) Products were prepared for identifying whether a test sample was infected with one or more of the 29 respiratory pathogens.
The invention also protects the application of the Taqman low-density microfluidic chip, which is any one of the following (a 1) to (a 6):
(a1) Identifying 29 respiratory pathogens;
(a2) Preparing products for identifying and identifying 29 respiratory pathogens;
(a3) Identifying whether the pathogen to be detected is one of 29 respiratory tract pathogens;
(a4) Preparing a product for identifying whether the pathogen to be detected is one of 29 respiratory pathogens;
(a5) Identifying whether the sample to be tested is infected with one or more of 29 respiratory pathogens;
(a6) Products were prepared for identifying whether a test sample was infected with one or more of the 29 respiratory pathogens.
The invention also protects a kit containing the Taqman low-density microfluidic chip; the application of the kit is (b 1) or (b 2) or (b 3):
(b1) Identifying 29 respiratory pathogens;
(b2) Identifying whether the pathogen to be detected is one of 29 respiratory tract pathogens;
(b3) And (3) identifying whether the sample to be tested is infected with one or more of 29 respiratory pathogens.
The kit also comprises other reagents for detecting 29 respiratory pathogens by using TAC, and specifically can be reagents in a real-time quantitative One-Step reverse transcription kit, such as 2 xQuant One Step Probe qRT-PCR master Mix, hotMaster Taq polymerase and QuantRTase.
Any one of the 29 respiratory pathogens influenza A virus, influenza B virus, parainfluenza virus1, parainfluenza virus2, parainfluenza virus3, human metapneumovirus A, human metapneumovirus B, rhinovirus, enterovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B, mycoplasma pneumoniae, chlamydia pneumoniae, streptococcus pneumoniae, tubercle bacillus, human bocavirus, measles virus, rubella virus, mumps virus, rickettsia, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, human coronavirus KU1, legionella pneumophila, haemophilus influenzae, bordetella pertussis I and Bordetella pertussis II.
Any one of the above-mentioned samples to be tested may specifically be nasopharyngeal swab samples.
The invention provides a Taqman low-density microfluidic chip (TAC) technical system for evaluating and optimizing 29 respiratory pathogens, aiming at the Taqman low-density microfluidic chip (TAC) for detecting 29 respiratory pathogens, so that the detection level of the Taqman low-density microfluidic chip (TAC) technical system can reach: the lowest detection limit is 10-100 copy/reaction; no cross reaction with other related pathogens and human genome; the coefficient of variation of repeatability is not more than 3%, and the coefficient of variation of stability is not more than 10%. Compared with a clinically common detection method, namely a gold standard, the sensitivity of the Taqman low-density microfluidic chip (TAC) detection technology for 29 respiratory pathogens is 95.2%, the specificity is 96.6%, and the consistency coefficient is 0.7-1.0. The Taqman low-density microfluidic chip (TAC) detection technology for 29 respiratory pathogens, which is established by the invention, fully utilizes the advantages of MGB probe detection, overcomes the defect of multiple detection of the traditional respiratory pathogens, can realize multi-target detection of the 29 respiratory pathogens on one sample within two hours, and has good sensitivity and specificity. The invention provides a rapid, accurate and high-flux technical means for monitoring and outbreak investigation of respiratory diseases in China.
Drawings
FIG. 1 shows TAC typesetting.
FIG. 2 is a view showing a TAC loading pattern.
Detailed Description
The following examples are intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set up and the results averaged.
The 29 respiratory pathogens tested by the present invention include: INF-A (influenzSub>A A Virus, influenzSub>A A), INF-B (influenzSub>A B Virus ), PIV-1 (parainfluenzSub>A Virus type 1, parainfluenzSub>A Virus 1), PIV-2 (parainfluenzSub>A Virus type 2, parainfluenzSub>A Virus 2), PIV-3 (parainfluenzSub>A Virus type 3, parainfluenzSub>A Virus 3), HMPV-A (Human metapneumovirus type A, human metapneumovirus A), HMPV-B (Human metapneumovirus type B, human metavirus B), HRV (Rhinovirus), HEV (Enterovirus, human Enterovirus), HADV (adenovirus, human Pan-adenovirus), RSV-Sub>A (Respiratory syncytial Virus Sub>A), RSV-B (Respiratory syncytial Virus B), m.pneumo (mycoplasmSub>A pneumoniae, mycoplasmSub>A pneumonie), C.pneumo (ChlamydiSub>A pneumoniae), S.pneumo (Streptococcus pneumoniae), MT (Mycobacterium tuberculosis), HBOV (Human bocavirus), MV (Measles Virus), RV (rubellSub>A Virus), MPV (Mumps Virus), CB (RickettsiSub>A, coxiellSub>A burnetii), HCoV-229E (Human coronavirus 22E, humancotoronius 229E), HCoV-NL63 (Human coronavirus NL63, humancotoronius NL 63), HCoV-OC43 (Human coronavirus 2243, humancoonius 2243), HK 1-HK 1, and Human pneumovirus HK 1, pan-legioniella), h.influ (Haemophilus influenzae, haemophilus influenza), b.pert I (Bordetella pertussis I, bordetella pertussis target I), and b.pert II (Bordetella pertussis II, bordetella pertussis target II).
Example 1 primer Probe design for detection of 29 respiratory pathogens
Primer probes for the detection of 29 respiratory pathogens are shown in table 1.
TABLE 1
Note: the degenerate base Y represents C or T; the degenerate base R represents A or G; the degenerate base S represents G or C; the degenerate base K represents G or T. Note: f represents an upstream primer; r represents a downstream primer; p represents an MGB probe. The 5 'end of the probe is marked with FAM fluorescent group, and the 3' end is marked with MGB group. In each primer probe combination, the molar ratio of the upstream primer to the downstream primer to the probe is 1.
In the invention, the single-plex real-time quantitative PCR adopts RNP as an internal reference gene, and the TAC detection adopts RNP and IPCO as internal reference genes. Primers and probes for detecting IPCO were designed and synthesized by ABI. Primers and probes for detecting RNP were as follows:
f:5 'AGATTTGGACCTGCGAGCG-containing 3' (SEQ ID NO: 88);
r:5 'GAGCGGCTGTCTCCACAAGTT-3' (SEQ ID NO: 89);
p:5'-CTGAAGGCTCTGCGC-3' (SEQ ID NO: 90).
Example 2 preparation of Positive plasmid Standard
Positive plasmid standards corresponding to each pathogen were prepared for 29 pathogens.
The desired fragment was inserted into the Cloning vector pEASY-Blunt zero (PEASY-Blunt zero Cloning Kit, all-round gold, lot No. CB 501) to obtain a positive plasmid standard (which had been sequence-verified).
The corresponding fragments of interest for each pathogen are shown in table 2.
TABLE 2
| Pathogens | GenBank | Number of bits |
| INF-A (influenzSub>A A virus) | MK995729.1 | 171-276 |
| INF-B (influenza B virus) | MK999133.1 | 657-747 |
| PIV-1 (parainfluenza virus type 1) | MH684390.1 | 778-859 |
| PIV-2 (parainfluenza virus type 2) | MH892406.1 | 7463-7619 |
| PIV-3 (parainfluenza virus type 3) | KT765995.1 | 291-549 |
| RSV-A (respiratory syncytial virus A type) | MH760599.1 | 1083-1166 |
| RSV-B (respiratory syncytial virus B type) | MK749916.1 | 1275-1377 |
| HMPV-A (human metapneumovirus A type) | MH428626.1 | 96-246 |
| HMPV-B (human metapneumovirus B type) | KY474534.1 | 442-522 |
| HRV (rhinovirus) | MH899592.1 | 138-345 |
| HADV (adenovirus) | MF962521.1 | 3-132 |
| HBOV (human bocavirus) | MK034749.1 | 2548-2628 |
| HEV (Enterovirus) | MH933857.1 | 325-470 |
| MV (measles virus) | MK161348.1 | 2005-2190 |
| RV (rubella virus) | LC466970.1 | 86-171 |
| HCoV-229E (human coronavirus 229E) | MF542265.1 | 25706-25782 |
| HCoV-NL63 (human coronavirus NL 63) | MG428707.1 | 26809-26927 |
| HCoV-OC43 (human coronavirus OC 43) | MK787437.1 | 438-513 |
| HCoV-HKU1 (human coronavirus KU 1) | LC315650.1 | 6053-6203 |
| MPV (mumps virus) | MK161348.1 | 2005-2190 |
| CB (rickettsia) | CP040059.1 | 1117266-1117162 |
| C.pneumo (chlamydia pneumoniae) | LN849043.1 | 132665-132830 |
| M. pneumo (mycoplasma pneumoniae) | LR214945.1 | 634494-634409 |
| L.pneu (legionella pneumophila) | LR134332.1 | 650170-650435 |
| S.pneumo (streptococcus pneumoniae) | LR536845.1 | 1841628-1841554 |
| Influ (Haemophilus influenzae) | CP031254.1 | 161576-161691 |
| MT (tubercle bacillus) | CP039850.1 | 3054551-3054389 |
| pertI (Bordetella pertussis I) | LR590467.1 | 4099514-4099579 |
| pertII (Bordetella pertussis II) | LR590480.1 | 108293-108236 |
Example 3 Single real-time quantitative PCR preliminary evaluation of primer probes
1. Clinical isolate strain detection
The strains to be tested: 29 clinically isolated pathogen strains.
CB (Rickettsia, coxiella burneti) is described in the literature: he Zen Sun will be in Yonghui et al, cloning and identification of Kirginia Belgium Jiuli strain II phase mutant [ J ] biotechnological communication, v.30 (2): 170-174. The public can be obtained from military medical research institute of national liberation military science institute.
HEV (Enterovirus, human Enterovirus) is described in the literature: voying, 2012-2014, chongqing Children respiratory virus infection epidemic characteristic analysis [ D ]. Military medical science institute of people's liberation army in China, 2015. The public can be obtained from military medical research institute of people's liberation army science institute.
MV (Measles Virus) is described in the literature: the design of probes specific for morbillivirus species is known from Zhu Guang, liu Bing, li bin, et al.
RV (rubella virus) is described in the literature: wang-Ching, daffy Poisson, zhangong, et al. RT-Se minimal-PCR for detection of rubella Virus [ J ]. J.Zhonghua journal of microbiology and immunology, 1995 (3). The public is available from the military medical institute of the national liberation force academy of sciences.
Pert I (Bordetella pertussis I Bordetella pertussis target I)/b.pert II (Bordetella pertussis II) are described in the literature: xugluhua, research on molecular identification of Bordetella pertussis, genomic polymorphism and micro-evolution [ D ].2011 the public can be obtained from the military medical research institute of the national military academy of people's liberation army.
M.pneumo (Mycoplasma pneumoniae), c.pneumo (Chlamydia pneumoniae) are described in the literature: wangfei, liyanwei, huang Xiaolan, et al, research and development and application of a reagent for clinical serological detection of Chlamydia pneumoniae and Mycoplasma pneumoniae [ J ]. Marker immunoassay and clinic, 2018 (5). The public can be obtained from military medical research institute of military academy of sciences of the people's liberation military.
Pneumo (Chlamydia pneumoniae) s.pneumo (Streptococcus pneumoniae), h.influ (Haemophilus influenzae) are described in literature: zhao C, wang X, zhang C, et al.development of a TaqMan Array card to target 21 scheduled pathologies [ J ]. BMC infectives Diseases,2019,19 (1).
MT (Mycobacterium tuberculosis) is described in the literature: lizhulin, xylonite, liu Ri Hui, et al, jilin province 110 Mycobacterium tuberculosis clinical isolates MLVA genotyping [ J ]. Jilin university Commission (medical edition), 2013,39 (2): 308-312. The public is available from military medical institute of national liberation force military academy.
L. pneu (Legionella pneumophila, pan-Legionella) is described in the literature: wanli, juhong, dianqin, detection of Legionella pneumophila colloidal gold immunochromatography establishment [ J ] military medicine, 2008,32 (1): 55-57. Public available from the military medical research institute of national institute of military sciences.
The remaining strains are described in: the public can be obtained from the military medical research institute of the national liberation military academy of sciences.
MPV (Mumps virus): the military medical research institute of the military science institute of the people's liberation army of China.
For each pathogen strain, single real-time quantitative PCR detection was performed using the strain cDNA as template and the primers and probes corresponding to this pathogen strain in Table 1.
Reaction system: 2 μ L of template, 12.5 μ L of 2 XQuant One Step Probe qRT-PCR master Mix, 1 μ L of HotMaster Taq polymerase, 0.5 μ L of QuantRTase, 0.5 μ L of Probe, 0.5 μ L of upstream and downstream primers, and 7.5 μ L of deionized water. The concentration of the probe in the reaction system was 200nmol, and the concentrations of the upstream and downstream primers in the reaction system were both 400nmol.
The reagents in the reaction system are all from Tiangen Biochemical technology Co., ltd (TIANGEN) in China: real-time quantitative One-Step reverse transcription Kit (Quant One Step qRT-PCR Kit (Probe).
After the reaction system is prepared, centrifuging at 1500r/s for 1min.
Reaction procedure: reverse transcription is carried out for 30min at 50 ℃, and pre-denaturation is carried out for 3min at 92 ℃;40 cycles of 92 ℃ denaturation 10seconds,62 ℃ annealing 20seconds; extension of 20seconds at 68 ℃ (where the fluorescence signal is collected).
CT values less than or equal to 38 are positive, and conversely, the result is negative. The results are shown in Table 3.
TABLE 3
The results show that the experimental results are all positive and have no cross reaction.
2. Positive standard plasmid detection
A sample to be detected: 29 positive standard plasmids prepared in example 2.
1. For each positive standard plasmid, configuration 10 7 copy/μL、10 6 copy/μL、10 5 copy/μL、10 4 copy/μL、10 3 copy/. Mu.L total 5 concentration gradients of test solution, each concentration setting 2 replicates. The primers and probes corresponding to the positive standard plasmid in Table 1 are adopted to carry out single real-time quantitative PCR detection, so that a standard curve is obtained, and the linearity and the amplification efficiency are obtained. The PCR reaction system and the reaction program are the same as the first step. The test was repeated 3 times to evaluate the reproducibility.
2. For each positive standard plasmid, from 10 2 cop/. Mu.L was diluted 5-fold, two concentration gradients were serially diluted, and 2 replicates were set for each concentration. The primers and probes corresponding to the positive standard plasmids in table 1 were used for single-plex real-time quantitative PCR detection, and the lowest detection Limit (LOD) was calculated.
The results are shown in Table 4. The result shows that 29 respiratory tract pathogen primer probes are evaluated by using the constructed positive standard: the amplification efficiency is more than 90 percent, and the lowest detection limit is less than 10 copy/mu L.
TABLE 4
Example 4 TAC detection evaluation of primer Probe
12 common respiratory pathogens (shown in Table 6) were selected and Applied biosystems, USA, were assigned to immobilize primers and probes for detection of 12 pathogens shown in Table 1 on a Taqman Array Card-dedicated reaction plate.
1. Evaluation of amplification efficiency and Linearity
For each respiratory pathogen, 3 positive standards were mixed at the same concentration, i.e., at the same order of magnitude, from 10 per TAC plate 7 copy/μl-10 4 copy/. Mu.l total 4 concentration gradients, each concentration gradient set for 2 replicates, constructing a standard curve, applying Applied BiosyFrom stems Ltd
The Universal Master Mix II was subjected to system construction and PCR reaction (Table 5), and the TAC amplification efficiency and the minimum detection limit evaluation were evaluated.
2. Evaluation of reproducibility and stability of the System
For each respiratory pathogen, a low concentration of 10 was taken 2 The copy/. Mu.L positive standard substance is repeatedly tested for three times in the same plate and different plates, and the repeatability and stability of the coefficient of variation evaluation system, namely CV, are calculated
The value is obtained.
3. Minimum detection Limit (LOD) of evaluation System
For each respiratory pathogen, a low concentration of 10 was taken 2 copy/. Mu.L positive standard from 10 2 cop/. Mu.L was diluted 5-fold, serially with two concentration gradients, each concentration set to 4 replicates, at least 3 of which showed an amplification concentration, defined as the lowest detection Limit (LOD).
TABLE 5
The results are shown in Table 6.
TABLE 6
Example 5 clinical sample testing
A sample to be tested: clinical samples containing 29 pathogens in table 1: one part is from subsidiary 307 hospital respiratory medicine and clinical laboratory of military medical academy of sciences; the other part is from the inspection department of the central control center for disease prevention and control in the cisterm of Beijing. 736 nasopharyngeal swab samples were collected from patients with suspected respiratory symptoms at 6 months-2015 5 months in 2013, 352 positive samples and 384 negative samples (with informed consent). The samples were stored in 1ml of sample solution and stored at-80 ℃ for future use.
The positive sample is a clinical sample (positive if infected by one of 29 pathogens) which is clinically detected to be positive by the existing traditional gold standard detection method (comprising cell culture, bacterial culture or single-fold real-time quantitative PCR) in the prior art. The negative sample is a clinical sample which is clinically detected by a traditional method and has no pathogen detected. The conventional laboratory tests were conducted by the subsidiary 307 hospital of the military medical academy of sciences providing samples and the centers for control of disease prevention in the cis-district of Beijing city, respectively.
The detection primers and probes for 29 pathogens shown in Table 1 were immobilized on a Taqman Array Card-dedicated reaction plate, applied biosystems, USA.
The TAC layout is shown in table 7 and fig. 1. The TAC loading pattern is shown in FIG. 2.
TABLE 7
| 1 | INF-A | INF-A | |
| 2 | INF-B | INF-B | |
| 3 | PIV-1 | PIV-1 | |
| 4 | PIV-2 | PIV-2 | |
| 5 | PIV-3 | PIV-3 | |
| 6 | HMPV-A | HMPV-A | |
| 7 | HMPV-B | HMPV-B | |
| 8 | HRV | HRV | |
| 9 | | HEV | |
| 10 | HADV | HADV | |
| 11 | IPCO | RNP | |
| 12 | RSV-A | RSV-A | |
| 13 | RSV-B | RSV-B | |
| 14 | M.pneumoniae | M.pneumoniae | |
| 15 | C.pneumoniae | C.pneumoniae | |
| 16 | S.pneumoniae | S.pneumoniae | |
| 17 | M.tuberculosis | M.tuberculosis | |
| 18 | HBOV | HBOV | |
| 19 | MV | RV | |
| 20 | MPV | C.burnetii | |
| 21 | HCoV-OC43 | HCoV-229E | |
| 22 | HCoV-NL63 | HCoV-HKU1 | |
| 23 | L.pneumophila | H.influenzae | |
| 24 | B.pertussis targetI | B.pertussis targetII |
And applying a one-step method reagent to construct a TAC reaction system, and detecting 352 parts of collected clinical positive samples and 384 parts of collected negative samples. Negative control: RNase-Free ddH 2 O; positive control: 10 2 cop/. Mu.L of positive standard.
The one-step reagent reaction system and reaction conditions are shown in Table 8.
TABLE 8
And (4) judging the standard: the positive and negative controls are normal and pollution-free during each amplification; the internal reference is normal each time; the cutoff value is set to 36.CT value less than 36, positive sample; CT values 36-38 are suspected cases; CT values greater than 38 are negative samples.
A clinical traditional detection method is used as a gold standard to evaluate the sensitivity and specificity of the TAC method. And (5) calculating the consistency detection result, namely the Kappa value, by using statistical software sps 17.0. Judgment criteria for Kappa value: <0,por; 0 to 0.20,light; 0.21 to 0.40, fair;0.41 to 0.60, moderate;0.61-0.80, substantaal; and 0.81-1.00, almost perfect aggregate, P < -0.05 are statistically significant.
The TAC detection effect compared to the gold standard method is shown in Table 9. Compared with the gold standard detection result, the overall sensitivity of the TAC is 95.2%, and the overall specificity is 96.6%.
A comparison of the TAC detection technique with the gold standard method is shown in table 10. The result shows that the detection consistency is better.
TABLE 9
The same clinical specimen was also tested for co-infection with two or more pathogens, with co-infected pathogens present in 24 samples (table 11).
In order to further evaluate the accuracy of the TAC detection technology, 16 gold standard considered positive samples were randomly extracted from the detection samples, and a first-generation sequencing was performed, wherein 15 of the 16 sequenced samples are consistent with the TAC detection result, and only 1 coronavirus sample is inconsistent with the detection result (Table 12).
TABLE 11
TABLE 12
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
<120> method for detecting 29 respiratory pathogens by using Taqman low-density microfluidic chip technology
<160> 90
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (5)..(5)
<223> r=a or g
<400> 1
gaccratcct gtcacctctg ac 22
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (9)..(9)
<223> y=c or t
<220>
<221> misc_feature
<222> (18)..(18)
<223> k=g or t
<400> 2
agggcattyt ggacaaakcg tcta 24
<210> 3
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgcagtcctc gctcact 17
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (5)..(5)
<223> y=c or t
<400> 4
tgaaygatgt ctgtttccaa agatc 25
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (5)..(5)
<223> s=g or c
<220>
<221> misc_feature
<222> (15)..(15)
<223> y=c or t
<400> 5
gggastgtgc ttccygcaaa 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aaagagttgg acttgaccct 20
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tgatttaaac ccggtaattt ctcat 25
<210> 8
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccttgttcct gcagctatta caga 24
<210> 9
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
acgacaacag gaaatc 16
<210> 10
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aggactatga aaaccattta cctaagtga 29
<210> 11
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
aagcaagtct cagttcagct agatca 26
<210> 12
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tgttcagtca ctgctatac 19
<210> 13
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aaaagttgat gaaagatcag attatgcat 29
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ccgggacacc cagttgtg 18
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aaaggcaaaa taatatttct c 21
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
agatcaactt ctgtcatcca gcaa 24
<210> 17
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ttctgcacat cataattagg agtatcaat 29
<210> 18
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cggagcacag gagat 15
<210> 19
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aagatgcaaa tcataaattc acagga 26
<210> 20
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tgatatccag catctttaag tatctttata gtg 33
<210> 21
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ctggacatag catataac 18
<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
agagatgtag gcaccacaac tgc 23
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ctgatcctag agccgtgcaa a 21
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ttcatcattg cagcaaga 18
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
acaatggcaa ctttgcttaa agaa 24
<210> 26
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gattataggt gtgtctggtg ctgaa 25
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
atattccaca aaatcagagg c 21
<210> 28
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ttccagcctg cgtggc 16
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gaaacacgga cacccaaagt agtc 24
<210> 30
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (11)..(11)
<223> y=c or t
<400> 30
cccctgaatg yggc 14
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cccagtggtc ttacatgcac at 22
<210> 32
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gccacggtgg ggtttctaa 19
<210> 33
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ccgggtctgg tgcag 15
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
agaggctcgg gctcatatca 20
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cacttggtct gaggtcttcg aa 22
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (7)..(7)
<223> r=a or g
<400> 36
caatcarcca cctatcgtct 20
<210> 37
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (5)..(5)
<223> y=c or t
<220>
<221> misc_feature
<222> (12)..(12)
<223> y=c or t
<400> 37
ggtgygaaga gyctattgag c 21
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
acggacaccc aaagtagtcg 20
<210> 39
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tccggcccct gaat 14
<210> 40
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gcaattggat caactgaagg c 21
<210> 41
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
agagtcagca tcttggattc cct 23
<210> 42
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
acagcggtga agcg 14
<210> 43
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
acgccgcacg gacaact 17
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tgttggttgc cggtgtaatt c 21
<210> 45
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
aggtcccgcc cgac 14
<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
cagtcaaatg ggctgatgca 20
<210> 47
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
caaagggcta taaagagaat aaggtattct 30
<210> 48
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
aacgtggtcg tcaggg 16
<210> 49
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gcgtgttcct accagagagg aa 22
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
gctgtggaaa acctttggca 20
<210> 51
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tgctttggtc ctcgtgat 18
<210> 52
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
cgatgaggct attccgacta ggt 23
<210> 53
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
ccttcctgag ccttcaatat agtaacc 27
<210> 54
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tggcacggta ctcc 14
<210> 55
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (14)..(14)
<223> y=c or t
<400> 55
agggatccta ctaytcaaga agctatcc 28
<210> 56
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
atgaacgatt attgggtcca cg 22
<210> 57
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
cgcctggtac gattt 15
<210> 58
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
gcaattggat caactgaagg c 21
<210> 59
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
agagtcagca tcttggattc cct 23
<210> 60
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
acagcggtga agcg 14
<210> 61
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
aatttggagc aaagccctta ga 22
<210> 62
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gtaaaaaggc gtcggcaata ac 22
<210> 63
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
accctggcat gtct 14
<210> 64
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
cgtggagcct tatgggaatg 20
<210> 65
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cgtctgttgg caagggga 18
<210> 66
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
cagtccaaac ctaaagtt 18
<210> 67
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
gcagttgctg gcgctaagtt 20
<210> 68
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
aagcgaggta cggtagcggt at 22
<210> 69
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
tggtagggaa ctcgtttta 19
<210> 70
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
gtactaattg gctgattgtc ttgacc 26
<210> 71
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
cctggcgatg acctactttc g 21
<210> 72
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
actctttacc aaacctg 17
<210> 73
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
acgcaatcta gcagatgaag ca 22
<210> 74
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
tcgtgcgttt taattccagc t 21
<210> 75
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
aacgcttgat acagggag 18
<210> 76
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
ggacaaacat cacaagcggt ta 22
<210> 77
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
tgcggtagtg ttagaaaatg gtattatg 28
<210> 78
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
ttgtagtatt gatacgcttt gt 22
<210> 79
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ggctgtgggt agcagacc 18
<210> 80
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
cgggtccaga tggcttg 17
<210> 81
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
acctgggcag ggtt 14
<210> 82
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
caaggccgaa cgcttcat 18
<210> 83
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
gagttctggt aggtgtgagc gtaa 24
<210> 84
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
ccttgcgtga gtggg 15
<210> 85
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
gccgccagct cgtacttc 18
<210> 86
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
ggatacggcc ggcattg 17
<210> 87
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
cgtcgacact tatggcga 18
<210> 88
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
agatttggac ctgcgagcg 19
<210> 89
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
gagcggctgt ctccacaagt 20
<210> 90
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
ctgaaggctc tgcgc 15
Claims (9)
1. The primer probe set comprises 29 primer probe sets;
the primer probe combination 1 consists of primers shown in sequences 1 and 2 of a sequence table and a probe shown in a sequence 3;
the primer probe combination 2 consists of primers shown in sequences 4 and 5 of a sequence table and a probe shown in a sequence 6;
the primer probe combination 3 consists of primers shown in sequences 7 and 8 of a sequence table and a probe shown in a sequence 9;
the primer probe combination 4 consists of primers shown in sequences 10 and 11 of a sequence table and a probe shown in a sequence 12;
the primer probe combination 5 consists of primers shown in sequences 13 and 14 of a sequence table and a probe shown in a sequence 15;
the primer probe combination 6 consists of primers shown in sequences 16 and 17 of the sequence table and a probe shown in a sequence 18;
the primer probe combination 7 consists of primers shown in sequences 19 and 20 of a sequence table and a probe shown in a sequence 21;
the primer probe combination 8 consists of primers shown in sequences 22 and 23 of the sequence table and a probe shown in a sequence 24;
the primer probe combination 9 consists of primers shown in sequences 25 and 26 of the sequence table and a probe shown in a sequence 27;
the primer probe combination 10 consists of primers shown in sequences 28 and 29 of a sequence table and a probe shown in a sequence 30;
the primer probe combination 11 consists of primers shown in sequences 31 and 32 of a sequence table and a probe shown in a sequence 33;
the primer probe combination 12 consists of primers shown in sequences 34 and 35 of a sequence table and a probe shown in a sequence 36;
the primer probe combination 13 consists of primers shown in sequences 37 and 38 of the sequence table and a probe shown in a sequence 39;
the primer probe combination 14 consists of primers shown in sequences 40 and 41 of a sequence table and a probe shown in a sequence 42;
the primer probe combination 15 consists of primers shown in sequences 43 and 44 of a sequence table and a probe shown in a sequence 45;
the primer probe combination 16 consists of primers shown in sequences 46 and 47 of the sequence table and a probe shown in a sequence 48;
the primer probe combination 17 consists of primers shown in sequences 49 and 50 of the sequence table and a probe shown in a sequence 51;
the primer probe combination 18 consists of primers shown in sequences 52 and 53 of a sequence table and a probe shown in a sequence 54;
the primer probe combination 19 consists of primers shown in sequences 55 and 56 of a sequence table and a probe shown in a sequence 57;
the primer probe combination 20 consists of primers shown in sequences 58 and 59 of a sequence table and a probe shown in a sequence 60;
the primer probe combination 21 consists of primers shown in sequences 61 and 62 of a sequence table and a probe shown in a sequence 63;
the primer probe combination 22 consists of primers shown in sequences 64 and 65 of a sequence table and a probe shown in a sequence 66;
the primer probe combination 23 consists of primers shown in sequences 67 and 68 of the sequence table and a probe shown in a sequence 69;
the primer probe combination 24 consists of primers shown in sequences 70 and 71 of a sequence table and a probe shown in a sequence 72;
the primer probe combination 25 consists of primers shown in sequences 73 and 74 of the sequence table and a probe shown in a sequence 75;
the primer probe combination 26 consists of primers shown in sequences 76 and 77 of the sequence table and a probe shown in a sequence 78;
the primer probe combination 27 consists of primers shown in sequences 79 and 80 of a sequence table and a probe shown in a sequence 81;
the primer probe combination 28 consists of primers shown in sequences 82 and 83 of the sequence table and a probe shown in a sequence 84;
the primer probe combination 29 consists of primers shown in sequences 85 and 86 of the sequence table and a probe shown in a sequence 87.
2. The set of primer probes of claim 1, wherein:
the set of primer probes further comprises a primer probe combination 30;
the primer probe combination 30 consists of primers shown in sequences 88 and 89 of the sequence table and a probe shown in a sequence 90.
3. The set of primer probes according to claim 1 or 2, characterized in that: the probe is an MGB probe; one end of the probe is marked by a fluorescent group, and the other end of the probe is marked by an MGB group.
4. The set of primer probes according to claim 1 or 2, characterized in that: in each primer probe combination, the molar ratio of the upstream primer to the downstream primer to the probe is 1.
5. Use of the primer-set probe of any one of claims 1 to 4, which is any one of the following (a 1) to (a 5):
(a1) Identifying 29 respiratory pathogens; the use is for non-disease diagnostic and therapeutic purposes;
(a2) Preparing a product for identifying 29 respiratory pathogens;
(a3) Identifying whether the pathogen to be detected is one of 29 respiratory pathogens; the use is for non-disease diagnostic and therapeutic purposes;
(a4) Preparing a product for identifying whether the pathogen to be tested is one of 29 respiratory pathogens;
(a5) Preparing a product for identifying whether a test sample is infected with one or more of the 29 respiratory pathogens;
the 29 respiratory pathogens are influenza A virus, influenza B virus, parainfluenza virus1, parainfluenza virus2, parainfluenza virus3, human metapneumovirus A, human metapneumovirus B, rhinovirus, enterovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B, mycoplasma pneumoniae, chlamydia pneumoniae, streptococcus pneumoniae, mycobacterium tuberculosis, human bocavirus, measles virus, rubella virus, mumps virus, rickettsia, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, human coronavirus KU1, legionella pneumophila, haemophila influenzae, bordetella pertussis I and bordetella pertussis II.
6. A kit comprising the primer probe set of any one of claims 1 to 4; the application of the kit is (b 1) or (b 2) or (b 3):
(b1) Identifying 29 respiratory pathogens;
(b2) Identifying whether the pathogen to be detected is one of 29 respiratory tract pathogens;
(b3) Identifying whether the sample to be tested is infected with one or more of 29 respiratory pathogens;
the 29 respiratory pathogens are influenza A virus, influenza B virus, parainfluenza virus1, parainfluenza virus2, parainfluenza virus3, human metapneumovirus A, human metapneumovirus B, rhinovirus, enterovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B, mycoplasma pneumoniae, chlamydia pneumoniae, streptococcus pneumoniae, mycobacterium tuberculosis, human bocavirus, measles virus, rubella virus, mumps virus, rickettsia, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, human coronavirus KU1, legionella pneumophila, haemophilus influenzae, bordetella pertussis I and bordetella pertussis II.
7. A Taqman low-density microfluidic chip; a set of primer probes according to any one of claims 1 to 4 is immobilized on the chip.
8. The use of the Taqman low density microfluidic chip of claim 7, which is any one of the following (a 1) to (a 5):
(a1) Identifying 29 respiratory pathogens; the use is for non-disease diagnostic and therapeutic purposes;
(a2) Preparing a product for identifying 29 respiratory pathogens;
(a3) Identifying whether the pathogen to be tested is one of 29 respiratory pathogens; the use is for non-disease diagnostic and therapeutic purposes; (a4) Preparing a product for identifying whether the pathogen to be tested is one of 29 respiratory pathogens;
(a5) Preparing a product for identifying whether a test sample is infected with one or more of the 29 respiratory pathogens;
the 29 respiratory pathogens are influenza A virus, influenza B virus, parainfluenza virus1, parainfluenza virus2, parainfluenza virus3, human metapneumovirus A, human metapneumovirus B, rhinovirus, enterovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B, mycoplasma pneumoniae, chlamydia pneumoniae, streptococcus pneumoniae, mycobacterium tuberculosis, human bocavirus, measles virus, rubella virus, mumps virus, rickettsia, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, human coronavirus KU1, legionella pneumophila, haemophila influenzae, bordetella pertussis I and bordetella pertussis II.
9. A kit comprising the Taqman low-density microfluidic chip of claim 7; the application of the kit is (b 1) or (b 2) or (b 3):
(b1) Identifying 29 respiratory pathogens;
(b2) Identifying whether the pathogen to be detected is one of 29 respiratory tract pathogens;
(b3) Identifying whether the sample to be tested is infected with one or more of 29 respiratory pathogens;
the 29 respiratory pathogens are influenza A virus, influenza B virus, parainfluenza virus1, parainfluenza virus2, parainfluenza virus3, human metapneumovirus A, human metapneumovirus B, rhinovirus, enterovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B, mycoplasma pneumoniae, chlamydia pneumoniae, streptococcus pneumoniae, mycobacterium tuberculosis, human bocavirus, measles virus, rubella virus, mumps virus, rickettsia, human coronavirus 229E, human coronavirus NL63, human coronavirus OC43, human coronavirus KU1, legionella pneumophila, haemophila influenzae, bordetella pertussis I and bordetella pertussis II.
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