Use of primers, probes and kits for the detection of respiratory pathogens

Abstract

The present application relates to biological detection, especially to the application of primer, probe and kit in the detection of respiratory pathogen, and provides a primer group, which comprises one or more of primer group 1 to primer group 12. The primer and probe provided by the present application have good specificity, and in combination with a real-time PCR detection method, can realize rapid, accurate and sensitive identification of FluA, FluB, RSV, MP, CPn, ADV, HPIV1, HPIV2, HPIV3, HMPV-A, HMPV-B and LP. The detection method of the present application is easy to operate, and has the characteristics of rapidity, simplicity and specificity. The present application provides an efficient detection method for individual patient symptom control and disease transmission prevention.

Description

Technical Field

[0001] This invention relates to biological detection, and more particularly to the application of primers, probes and kits in the detection of respiratory pathogens. Background Technology

[0002] Respiratory infections involve numerous pathogens and opportunistic microorganisms, including bacteria, viruses, mycoplasma, fungi, and parasites. However, the symptoms and signs they cause are generally similar, manifesting as cough, sputum production, sneezing, runny nose, fever, and difficulty breathing. Yet, the clinical treatments for different types of pathogens vary greatly. For example, viral pathogens require different types of antiviral drugs, while bacterial pathogens require even more diverse antibiotics. Because the risks posed by different types of pathogens vary significantly—some are self-limiting, while others can lead to serious complications and endanger life, and some can even cause the rapid spread and outbreak of severe respiratory infectious diseases—it is crucial to quickly identify the type of infecting pathogen in clinical diagnosis and treatment. This is essential for implementing specific treatment measures promptly, thereby reducing drug overuse, improving patient cure and survival rates, and reducing the social burden and expenditure on healthcare. It is also of great significance for the early detection and screening of pathogens causing emerging respiratory infectious diseases.

[0003] Therefore, PCR technology, which amplifies specific nucleic acid fragments of pathogens, plays a very important role. Its rapid, accurate, and highly sensitive detection characteristics have gradually made it an irreplaceable technical means, and many PCR reagents for the detection of single pathogens are now widely used in medical practice.

[0004] However, PCR reagents that detect single pathogens force doctors to rely on experience to start testing for the most likely pathogens and then systematically rule them out. This approach is not only time-consuming and costly, but it also exacerbates patient suffering and ultimately delays optimal treatment. Therefore, doctors often have to prescribe medication empirically without a clear etiological diagnosis, leading to drug abuse, increased drug toxicity and the likelihood of pathogen resistance, wasted medical resources, and reduced treatment effectiveness.

[0005] For many years, scholars both domestically and internationally have been dedicated to developing technologies for the simultaneous detection of multiple targets. However, few products using such technologies have yet achieved success. This is because the presence of multiple primer-probe pairs in a single reaction system can lead to unimaginable complexities. As is well known, all DNA consists of only four bases: A, T, C, and G. Therefore, the probability of three consecutive bases being identical to other sequences is relatively high. Furthermore, as the number of primers increases, the likelihood of overlapping or complementary sequences increases rapidly, becoming almost unavoidable. If several consecutive complementary bases exist between primers, they are highly likely to bind and amplify under the action of polymerase, resulting in a large number of nonspecific false-positive amplification products. Therefore, primers previously used for single-target detection are unsuitable for multiplex PCR.

[0006] The solution to the above problems is to carefully redesign primers and probes, making the differences between primer and probe sequences for each target as large as possible to avoid complementary regions. The differences in the composition of each primer and probe sequence inevitably lead to significant differences in GC / AT content, resulting in large differences in their melting temperatures (Tm values). However, due to the requirements of polymerase recognition ability and amplification specificity, the length of primers and probes is limited to a very small range. Therefore, the ability to adjust the Tm value is limited. Furthermore, the general theory of PCR suggests that better amplification efficiency and specificity are obtained when the annealing / extension temperature is close to the Tm value. Therefore, excessively large differences in Tm values ​​will lead to inconsistencies in the amplification efficiency and specificity of different targets in the same reaction system, and may even easily cause some targets to be missed while others show false positives. In addition, for many pathogens, there are numerous non-pathogenic bacteria that are very similar in species classification to them, both inside the human body and in the external environment. These two species share a large number of homologous sequences, which poses a challenge in selecting specific primers and probes. As the number of primers and probes required for multi-target detection increases, the difficulty of sequence selection becomes much greater than for single-target detection. The risk of false positives due to cross-reactions caused by similar microbial species also increases dramatically. Therefore, there is currently a lack of fluorescent quantitative PCR kits that can accurately and sensitively detect and differentiate multiple respiratory pathogens simultaneously. Summary of the Invention

[0007] In view of this, the present invention provides primers, probes, and kits for the detection of respiratory pathogens. The primers and probes provided by the present invention can simultaneously detect FluA, FluB, RSV, MP, CPn, ADV, HPIV1, HPIV2, HPIV3, HMPV-A, HMPV-B, and LP. These primers and probes have good specificity, and when combined with real-time PCR detection, they can achieve rapid, accurate, and sensitive identification of the above-mentioned respiratory pathogens, while reducing false positives.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] The present invention provides primer sets, including one or more of primer sets 1 to 12;

[0010] Primer set 1: The primer set used to amplify influenza A virus has the following characteristics:

[0011] (I) Nucleotide sequences as shown in SEQ ID NO:1 and SEQ ID NO:2; or

[0012] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0013] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0014] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0015] Primer set 2: Primers used for amplifying influenza B virus have the following characteristics:

[0016] (I) Nucleotide sequences as shown in SEQ ID NO:4 and SEQ ID NO:5; or

[0017] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0018] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0019] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0020] Primer set 3: Primers used for amplifying respiratory syncytial virus have the following characteristics:

[0021] (I) Nucleotide sequences as shown in SEQ ID NO:7 and SEQ ID NO:8; or

[0022] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0023] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0024] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0025] Primer set 4: Primers used for amplifying Mycoplasma pneumoniae have the following characteristics:

[0026] (I) Nucleotide sequences as shown in SEQ ID NO:10 and SEQ ID NO:11; or

[0027] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0028] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0029] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0030] Primer set 5: Primers used for amplifying Chlamydia pneumoniae have the following characteristics:

[0031] (I) Nucleotide sequences as shown in SEQ ID NO:13 and SEQ ID NO:14; or

[0032] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0033] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0034] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0035] Primer set 6: Primers used for amplifying adenovirus have the following characteristics:

[0036] (I) Nucleotide sequences as shown in SEQ ID NO:16 and SEQ ID NO:17; or

[0037] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0038] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0039] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0040] Primer set 7: Primers used for amplifying parainfluenza type 1 virus have the following characteristics:

[0041] (I) Nucleotide sequences as shown in SEQ ID NO:19 and SEQ ID NO:20; or

[0042] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0043] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0044] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0045] Primer set 8: Primers used for amplifying parainfluenza type 2 virus have the following characteristics:

[0046] (I) Nucleotide sequences as shown in SEQ ID NO:22 and SEQ ID NO:23; or

[0047] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0048] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0049] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0050] Primer set 9: Primers used for amplifying parainfluenza type 3 virus have the following characteristics:

[0051] (I) Nucleotide sequences as shown in SEQ ID NO:25 and SEQ ID NO:26; or

[0052] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0053] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0054] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0055] Primer set 10: Primers for amplifying human metapneumovirus type A have the following characteristics:

[0056] (I) Nucleotide sequences as shown in SEQ ID NO:28 and SEQ ID NO:29; or

[0057] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0058] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0059] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0060] Primer set 11: Primers for amplifying human metapneumovirus type B have the following characteristics:

[0061] (I) Nucleotide sequences as shown in SEQ ID NO:31 and SEQ ID NO:32; or

[0062] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0063] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0064] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0065] Primer set 12: Primers used for amplifying Legionella have the following characteristics:

[0066] (I) Nucleotide sequences as shown in SEQ ID NO:34 and SEQ ID NO:35; or

[0067] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0068] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0069] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequences described in (I), (II) or (III).

[0070] The present invention also provides a probe set, comprising one or more of probe set 1 to probe set 12;

[0071] Probe set 1: Probes for amplifying influenza A virus have the following characteristics:

[0072] (I) A nucleotide sequence as shown in SEQ ID NO:3; or

[0073] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0074] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0075] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0076] Probe set 2: Probes for amplifying influenza B virus have the following characteristics:

[0077] (I) A nucleotide sequence as shown in SEQ ID NO:6; or

[0078] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0079] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0080] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0081] Probe set 3: Probes for amplifying respiratory syncytial virus have the following characteristics:

[0082] (I) A nucleotide sequence as shown in SEQ ID NO:9; or

[0083] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0084] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0085] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0086] Probe set 4: Probes for amplifying Mycoplasma pneumoniae have the following characteristics:

[0087] (I) A nucleotide sequence as shown in SEQ ID NO:12; or

[0088] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0089] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0090] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0091] Probe set 5: Probes for amplifying Chlamydia pneumoniae have the following characteristics:

[0092] (I) A nucleotide sequence as shown in SEQ ID NO:15; or

[0093] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0094] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0095] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0096] Probe set 6: Probes for amplifying adenovirus have the following characteristics:

[0097] (I) A nucleotide sequence as shown in SEQ ID NO:18; or

[0098] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0099] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0100] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0101] Probe set 7: Probes for amplifying parainfluenza type 1 virus have the following characteristics:

[0102] (I) A nucleotide sequence as shown in SEQ ID NO:21; or

[0103] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0104] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0105] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0106] Probe set 8: Probes for amplifying parainfluenza type 2 virus have the following characteristics:

[0107] (I) A nucleotide sequence as shown in SEQ ID NO:24; or

[0108] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0109] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0110] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0111] Probe set 9: Probes for amplifying parainfluenza type 3 virus have the following characteristics:

[0112] (I) A nucleotide sequence as shown in SEQ ID NO:27; or

[0113] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0114] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0115] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0116] Probe set 10: Probes for amplifying human metapneumovirus type A have the following characteristics:

[0117] (I) A nucleotide sequence as shown in SEQ ID NO:30; or

[0118] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0119] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0120] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0121] Probe set 11: Probes for amplifying human metapneumovirus type B primers have the following characteristics:

[0122] (I) A nucleotide sequence as shown in SEQ ID NO:33; or

[0123] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0124] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0125] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in (I), (II) or (III); and / or

[0126] Probe set 12: Probes for amplifying Legionella have the following characteristics:

[0127] (I) A nucleotide sequence as shown in SEQ ID NO:36; or

[0128] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0129] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0130] (IV) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequences described in (I), (II) or (III).

[0131] In some embodiments of the present invention, the 5' end of the nucleotide sequence of the probe set is linked to a fluorescent reporter gene, and the 3' end is linked to a quencher gene.

[0132] In some embodiments of the present invention, the primers and probes are divided into primer-probe group A, primer-probe group B, primer-probe group C, and primer-probe group D. The 5' ends of the four probes in primer-probe group A are respectively connected to different fluorescent reporter groups; the 5' ends of the four probes in primer-probe group B are respectively connected to different fluorescent reporter groups; the 5' ends of the four probes in primer-probe group C are respectively connected to different fluorescent reporter groups; and the 5' ends of the three probes in primer-probe group D are respectively connected to different fluorescent reporter groups. Nine respiratory pathogens can be detected simultaneously in four tubes, enabling rapid, accurate, and complete detection of respiratory pathogens, which is of great significance for symptom control in individual patients and prevention of disease transmission.

[0133] The present invention also provides the application of the above-described primer set and / or probe set in the preparation of reagents and / or kits for detecting respiratory diseases.

[0134] The present invention also provides detection products comprising the above-described primer set and / or the above-described probe set, as well as acceptable adjuvants.

[0135] In some embodiments of the present invention, the testing product further includes:

[0136] The primers for the human 18S RNA internal standard have the nucleotide sequences shown in SEQ ID NO:37 and SEQ ID NO:38, and the probe for the human 18S RNA internal standard has the nucleotide sequence shown in SEQ ID NO:39; and / or

[0137] The primers for the human globin DNA internal standard have nucleotide sequences as shown in SEQ ID NO:40 and SEQ ID NO:41, and the probe for the human globin DNA has a nucleotide sequence as shown in SEQ ID NO:42.

[0138] In some embodiments of the present invention, the detection product includes the above-described primer set and / or a reaction solution such as the above-described probe set;

[0139] The above reaction solution includes: Tricine, KOAc, Tween20, glycerol, DMSO, betaine, dNTPs, Tth enzyme, manganese acetate, and water.

[0140] In some embodiments of the present invention, the addition of an appropriate concentration of DMSO to the reaction solution adjusts the thermal conductivity and surface tension of the reaction solution to ensure the homogeneity of the complex amplification reaction. Betaine, as a mild DNA denaturant, at this concentration, makes the Tm values ​​of these primers and probes tend to be consistent, thereby contributing to the coordinated amplification efficiency and specificity of multi-target detection under the same amplification temperature cycle. Furthermore, Tth enzymes possess both reverse transcription and DNA polymerization activities, and the conformation of their active sites responsible for nucleic acid recognition and replication is specific, resulting in differences in the activation effects of manganese, magnesium, and potassium ions on their activity; that is, the three have different effects on reverse transcription, DNA replication, and amplification specificity and efficiency. Therefore, in the present invention, their ratios and concentrations are specifically optimized to balance these characteristics in the aforementioned primer-probe combination. Simultaneously, the pH value of the reaction system also has different effects on the amplification specificity and efficiency of different primers and probes, and this has also been specifically optimized in the present invention to adapt it to the aforementioned primer-probe combination.

[0141] The present invention also provides a method for using the above-mentioned detection product, wherein the above-mentioned detection product is mixed with the sample respectively, and a fluorescent signal appears, indicating that the sample is positive.

[0142] In some embodiments of the present invention, the fluorescence detection channel is selected as follows:

[0143] In System 1, the FAM channel was selected to detect influenza A virus; the HEX channel was selected to detect human 18S RNA internal standard; the ROX channel was selected to detect influenza B virus; and the CY5 channel was selected to detect respiratory syncytial virus.

[0144] In System 2, the FAM channel was selected to detect Mycoplasma pneumoniae; the HEX channel was selected to detect human globin DNA internal standard; the ROX channel was selected to detect adenovirus; and the CY5 channel was selected to detect Chlamydia pneumoniae.

[0145] In System 3, the FAM channel was selected to detect parainfluenza type 1; the HEX channel was selected to detect human 18S RNA internal standard; the ROX channel was selected to detect parainfluenza type 3; and the CY5 channel was selected to detect parainfluenza type 2.

[0146] In System 4, the FAM channel was selected to detect human metapneumovirus type A; the HEX channel was selected to detect human 18S RNA internal standard; the ROX channel was selected to detect human metapneumovirus type B; and the CY5 channel was selected to detect Legionella.

[0147] The specific test results are as follows: the threshold is set to 3.

[0148] In reaction tube 1:

[0149] The probe channels shown in SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.9 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.39 was ≤35. The test result was reported as negative.

[0150] If the CT value of the probe channel shown in SEQ ID NO.3 is ≤36, it is reported as positive for influenza A virus.

[0151] If the CT value of the probe channel shown in SEQ ID NO.6 is ≤36, it is reported as positive for influenza B virus.

[0152] If the CT value of the probe channel shown in SEQ ID NO.9 is ≤36, it is reported as a positive respiratory syncytial virus (RSV) test.

[0153] In reaction tube 2:

[0154] The probe channels shown in SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.42 was ≤35. Therefore, the test result was reported as negative.

[0155] If the CT value of the probe channel shown in SEQ ID NO.12 is ≤36, it is reported as positive for Mycoplasma pneumoniae.

[0156] If the CT value of the probe channel shown in SEQ ID NO.15 is ≤36, it is reported as positive for Chlamydia pneumoniae.

[0157] If the CT value of the probe channel shown in SEQ ID NO.18 is ≤35, it is reported as adenovirus positive;

[0158] In reaction tube 3:

[0159] The probe channels shown in SEQ ID NO.21, SEQ ID NO.24, and SEQ ID NO.27 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.39 was ≤35. Therefore, the test result was reported as negative.

[0160] If the CT value of the probe channel shown in SEQ ID NO.21 is ≤35, it is reported as positive for parainfluenza virus type 1.

[0161] If the CT value of the probe channel shown in SEQ ID NO.24 is ≤35, it is reported as positive for parainfluenza virus type 2.

[0162] SEQ ID NO.27, the probe channel shown has a CT value ≤35, and is reported as positive for parainfluenza virus type 3;

[0163] In reaction tube 4:

[0164] The probe channels shown in SEQ ID NO.30, SEQ ID NO.33, and SEQ ID NO.36 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.39 was ≤35. The test result was reported as negative.

[0165] If the CT value of the probe channel shown in SEQ ID NO.30 is ≤36, it is reported as positive for human metapneumovirus type A.

[0166] SEQ ID NO.33, the probe channel shown has a CT value ≤36, and is reported as positive for human metapneumovirus type B;

[0167] If the CT value of the probe channel shown in SEQ ID NO.36 is ≤34, it is reported as Legionella positive.

[0168] If the CT value of the probe channel shown in SEQ ID NO. 39 or SEQ ID NO. 42 is ≥35, and either the negative control shows a CT value or a typical S amplification curve, or the positive control shows no CT value or no amplification curve, the test result is invalid. The cause should be identified and eliminated, and the test should be repeated.

[0169] In some embodiments of the present invention, the reaction system of the above-mentioned mixture used in the method includes: 20 μL of system A to D, 10 μL of system E, and 50 μL of sample;

[0170] The system A to D consisted of: Tricine 4 μL, KOAc 0.9 μL, Tween 200.1 μL, glycerol 1.1 μL, DMSO 3 μL, 5M betaine 2.3 μL, dNTPS 1 μL, rtTh enzyme 3.5 μL, the final concentration of the primer set was 0.3 μM, the final concentration of the probe set was 0.15 μM, and ultrapure water was added to a final volume of 20 μL.

[0171] System E consists of: 3.5 μL of 50 mM manganese acetate and ultrapure water to a final volume of 10 μL.

[0172] The present invention also provides the application of the above-described primer set, the above-described probe set, and / or the above-described detection product in the preparation of one or more products for detecting FluA, FluB, RSV, MP, CPn, ADV, HPIV1, HPIV2, HPIV3, HMPV-A, HMPV-BA, HMPV-A, HMPV-BB, and LP.

[0173] This invention provides the application of primers, probes, and kits in the detection of respiratory pathogens. The invention provides primer sets comprising one or more of primer sets 1 to 12, the primer set sequences of which are shown in Table 1.

[0174] The primers and probes provided by this invention have good specificity. Combined with the real-time PCR detection method, they can achieve rapid, accurate, and sensitive identification of FluA, FluB, RSV, MP, CPn, ADV, HPIV1, HPIV2, HPIV3, HMPV-A, HMPV-B, and LP. The detection method of this invention is easy to operate and has the characteristics of being rapid, simple, and specific. It provides an efficient detection method for symptom control in individual patients and prevention of disease transmission. Attached Figure Description

[0175] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0176] Figure 1 The amplification detection results of the positive sample in reaction tube 1 are shown;

[0177] Figure 2 The amplification detection results of the positive sample in reaction tube 2 are shown;

[0178] Figure 3 The amplification detection results of the positive sample in reaction tube 3 are shown;

[0179] Figure 4 The amplification detection results of the positive sample in reaction tube 4 are shown. Detailed Implementation

[0180] This invention discloses the application of primers, probes, and kits in the detection of respiratory pathogens.

[0181] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0182] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0183] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0184] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0185] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0186] All raw materials and reagents used in this invention are commercially available.

[0187] The present invention will be further illustrated below with reference to the embodiments:

[0188] Example 1: Primers, probes, and amplification sequences

[0189] The primer and probe sequences in the kit are shown in Table 1 below:

[0190] Table 1 Primer and probe sequences

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] Example 2: Detection process of the kit

[0197] The detection method of this invention is Real-time RT-PCR, and the Real-time RT-PCR reaction process is as follows:

[0198] (1) Reverse transcription: Under the action of reverse transcriptase, complementary cDNA is synthesized. The time and length depend on the length of the target nucleic acid and the base composition. The pre-denaturation temperature is generally 42℃~60℃, and the time and length depend on the length of the target nucleic acid and the base composition. The pre-denaturation temperature is generally 90℃~105℃, and the time is generally 10~20min.

[0199] (2) Pre-denaturation: The time and length depend on the length and base composition of the target nucleic acid. The temperature of pre-denaturation is generally 90℃~105℃, and the time is generally 2~10min. The purpose of pre-denaturation is to completely separate the double-stranded nucleic acid sequence into single strands.

[0200] (3) Denaturation, the temperature is generally 91℃~105℃, and the time is generally 10s~35s;

[0201] (4) Annealing, to anneal each primer to bind to the target sequence. The annealing temperature is usually 40℃~60℃, and the annealing time can be 10s~60s;

[0202] (5) Extension: The primer binds to the template and begins to synthesize a new DNA double strand. The extension temperature is generally 40℃~80℃ and the extension time can be 10s~1min.

[0203] The fluorescence detection channels selected in this invention are as follows:

[0204] (1) In System 1, the FAM channel was selected to detect influenza A virus; the HEX channel was selected to detect human 18S RNA internal standard; the ROX channel was selected to detect influenza B virus; and the CY5 channel was selected to detect respiratory syncytial virus.

[0205] (2) In System 2, the FAM channel was selected to detect Mycoplasma pneumoniae; the HEX channel was selected to detect human globin DNA internal standard; the ROX channel was selected to detect adenovirus; and the CY5 channel was selected to detect Chlamydia pneumoniae.

[0206] (3) In System 3, the FAM channel was selected to detect parainfluenza type 1; the HEX channel was selected to detect human 18S RNA internal standard; the ROX channel was selected to detect parainfluenza type 3; and the CY5 channel was selected to detect parainfluenza type 2.

[0207] (4) In System 4, the FAM channel was selected to detect human metapneumovirus type A; the HEX channel was selected to detect human 18S RNA internal standard; the ROX channel was selected to detect human metapneumovirus type B; and the CY5 channel was selected to detect Legionella.

[0208] The specific test results are as follows:

[0209] The threshold is set to 3, (1) in reaction tube 1:

[0210] The probe channels shown in SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.9 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.39 was ≤35. The test result was reported as negative.

[0211] If the CT value of the probe channel shown in SEQ ID NO.3 is ≤36, it is reported as positive for influenza A virus.

[0212] If the CT value of the probe channel shown in SEQ ID NO.6 is ≤36, it is reported as positive for influenza B virus.

[0213] If the CT value of the probe channel shown in SEQ ID NO.9 is ≤36, it is reported as a positive respiratory syncytial virus (RSV) test.

[0214] (2) In reaction tube 2:

[0215] The probe channels shown in SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO.18 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.42 was ≤35. Therefore, the test result was reported as negative.

[0216] If the CT value of the probe channel shown in SEQ ID NO.12 is ≤36, it is reported as positive for Mycoplasma pneumoniae.

[0217] If the CT value of the probe channel shown in SEQ ID NO.15 is ≤36, it is reported as positive for Chlamydia pneumoniae.

[0218] If the CT value of the probe channel shown in SEQ ID NO.18 is ≤35, it is reported as adenovirus positive;

[0219] (3) In reaction tube 3:

[0220] The probe channels shown in SEQ ID NO.21, SEQ ID NO.24, and SEQ ID NO.27 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.39 was ≤35. Therefore, the test result was reported as negative.

[0221] If the CT value of the probe channel shown in SEQ ID NO.21 is ≤35, it is reported as positive for parainfluenza virus type 1.

[0222] If the CT value of the probe channel shown in SEQ ID NO.24 is ≤35, it is reported as positive for parainfluenza virus type 2.

[0223] SEQ ID NO.27, the probe channel shown has a CT value ≤35, and is reported as positive for parainfluenza virus type 3;

[0224] (4) In reaction tube 4:

[0225] The probe channels shown in SEQ ID NO.30, SEQ ID NO.33, and SEQ ID NO.36 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.39 was ≤35. The test result was reported as negative.

[0226] If the CT value of the probe channel shown in SEQ ID NO.30 is ≤36, it is reported as positive for human metapneumovirus type A.

[0227] SEQ ID NO.33, the probe channel shown has a CT value ≤36, and is reported as positive for human metapneumovirus type B;

[0228] If the CT value of the probe channel shown in SEQ ID NO.36 is ≤34, it is reported as Legionella positive.

[0229] If the CT value of the probe channel shown in SEQ ID NO.39 or SEQ ID NO.42 is ≥35, and either the negative control shows a CT value or a typical S amplification curve, or the positive control shows no CT value or no amplification curve, the test result is invalid. The cause should be identified and eliminated, and the test should be repeated.

[0230] Example 3: Feasibility test of the reagent kit

[0231] Test it with any potentially interfering samples.

[0232] (1) The kit of the present invention was prepared by the method of Example 1;

[0233] (2) The positive samples were tested using the kit from Example 1. The results showed that all positive samples were positive. (See attached table for details.) Figures 1-4 ;

[0234] (3) The kit of Example 1 was used to detect positive nucleic acids of measles virus, mumps virus, rubella virus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, human parainfluenza virus type 4, and Candida albicans. The results showed no cross-reactivity. The results are shown in Table 2.

[0235] Table 2 Cross-reactivity test

[0236]

[0237]

[0238] The results showed that the kit of the present invention has good specificity for nine respiratory pathogens, and can specifically detect FluA, FluB, RSV, MP, CPn, ADV, HPIV1, HPIV2, HPIV3, HMPV-A, HMPV-B and LP, and there is no cross-reaction with other pathogens.

[0239] Example 4: Detection sensitivity, accuracy, and specificity of the kit

[0240] (1) Detection sensitivity of the kit of the present invention

[0241] For nine pathogens, samples of different concentrations were used to amplify nucleic acids using the real-time RT-PCR method with this detection kit.

[0242] RNA was extracted from each pathogen, the RNA template concentration was determined, and the RNA was diluted to 10⁻⁶ according to the specified ratio. -2 The initial concentration was ng / μL, which was then serially diluted 10-fold to obtain 10 ng / μL. -3 10 -4 10 -5 and 10 -6 Seven concentration gradients were used as templates for fluorescent PCR nucleic acid amplification according to the kit's sample addition method. The kit's detection results are shown in Table 3.

[0243] Table 3. Sensitivity tests for each pathogen

[0244]

[0245]

[0246] The results show that the primer-probe combination designed in this invention has high sensitivity. The detection sensitivity of the influenza A virus primer-probe reached 10. -5 ng / μL; the detection sensitivity of the influenza B virus primer probe reached 10 ng / μL. -5 ng / μL; the detection sensitivity of the respiratory syncytial virus primer probe reached 10 ng / μL. -5 ng / μL; the detection sensitivity of the Mycoplasma pneumoniae primer probe reached 10 ng / μL. -5 ng / μL; the detection sensitivity of the Chlamydia pneumoniae primer probe reached 10 ng / μL. -5 ng / μL; the detection sensitivity of the adenovirus primer probe reached 10 ng / μL. -4 ng / μL; the detection sensitivity of the parainfluenza virus type 1 primer probe reached 10 ng / μL. -4 ng / μL; the detection sensitivity of the parainfluenza virus type 2 primer probe reached 10 ng / μL. -4 ng / μL; the detection sensitivity of the parainfluenza virus type 3 primer probe reached 10 ng / μL. -4The detection sensitivity of the human metapneumovirus type A primer probe reached 10 ng / μL. -5 The detection sensitivity of the human metapneumovirus type B primer probe reached 10 ng / μL. -5 ng / μL; the detection sensitivity of the Legionella primer probe reached 10 ng / μL. -3 ng / μL.

[0247] (2) Detection accuracy and specificity of the kit of the present invention

[0248] Using the kit of this invention, 17 pathogens were detected, including 9 pathogens such as influenza A virus, influenza B virus, respiratory syncytial virus, mycoplasma pneumoniae, chlamydia pneumoniae, adenovirus, parainfluenza types 1, 2, and 3 (HPIV1, HPIV2, HPIV3), human metapneumovirus (HMPV-A, HMPV-B), Legionella, and 8 other pathogens such as measles virus, mumps virus, rubella virus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, human parainfluenza virus type 4, and Candida albicans.

[0249] Nucleic acid from each pathogen was extracted and set aside. Using the fluorescent PCR detection kit reaction system, the nucleic acid template of the sample to be tested was added, and the detection was performed using the recommended reaction procedure of the kit. The detection results are shown in Table 4.

[0250] Table 4. Accuracy and Specificity Tests for Each Pathogen

[0251]

[0252]

[0253] The results showed that nine pathogens produced values ​​in their respective fluorescent channels. Based on the fluorescence detection results, these were identified as influenza A virus, influenza B virus, respiratory syncytial virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, parainfluenza types 1, 2, and 3 (HPIV1, HPIV2, HPIV3), human metapneumovirus (HMPV-A, HMPV-B), and Legionella. No amplification curves or fluorescence signals were observed in the channels for measles virus, mumps virus, rubella virus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, human parainfluenza virus type 4, Candida albicans, and the negative control.

[0254] The above results indicate that the detection kit can detect nine pathogens with high sensitivity and specificity, and there is no cross-reaction between the pathogens. It can accurately and effectively detect nine pathogens simultaneously in a sample.

[0255] Example 5: Stability assessment of the reagent kit of the present invention

[0256] (1) FluA, FluB, RSV, MP, CPn, ADV, HPIV1, HPIV2, HPIV3, HMPV-A, HMPV-B, and LP nucleic acid detection were prepared by means of Example 1.

[0257] (2) Sample detection

[0258] The nucleic acids of each pathogen were added to nucleic acid detection reagent reaction tubes accelerated at 30°C for 7, 10, and 14 days, with two replicates for each type. At the same time, 50 μl of purified water was added to the detection solution as a negative control, and the detection was performed according to the detection method in Example 2.

[0259] (3) Results Analysis

[0260] The reagent kits prepared in Example 1 and the detection method in Example 2 were used to detect FluA, FluB, RSV, MP, CPn, ADV, HPIV1, HPIV2, HPIV3, HMPV-A, HMPV-B, and LP nucleic acid detection reagents transported at 2–8°C for 7 days and accelerated at 30°C for 7, 10, and 14 days. The specific data are shown in Table 5.

[0261] Table 5 Accelerated Stability Test

[0262] Acceleration (30℃) time (d) Detection status 7 Stable detection 10 Stable detection 14 Stable detection

[0263] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Zhengzhou Antu Bioengineering Co., Ltd. <120> Application of primers, probes and kits in the detection of respiratory pathogens <130> MP21032388 <160> 56 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty four <212> DNA <213> Artificial Sequence <400> 1 agaccaatcc tgtcacctct gact 24 <210> 2 <211> twenty four <212> DNA <213> Artificial Sequence <400> 2 agggcattyt ggacaaakcg tcta 24 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 acgctcaccg tgcccagtga gc 22 <210> 4 <211> twenty four <212> DNA <213> Artificial Sequence <400> 4 agaagacgga ggactaccac aaag 24 <210> 5 <211> twenty three <212> DNA <213> Artificial Sequence <400> 5 cttcatgaag gcaatctgct tca 23 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <400> 6 agaattgttg ttgattacat ggtgcaaa 28 <210> 7 <211> twenty four <212> DNA <213> Artificial Sequence <400> 7 agcttcacga aggctccaca taca 24 <210> 8 <211> twenty four <212> DNA <213> Artificial Sequence <400> 8 agcttcacga aggctccaca taca 24 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <400> 9 agcwgctgtc cagtacaatg ttctag 26 <210> 10 <211> twenty three <212> DNA <213> Artificial Sequence <400> 10 agtcggacca aaccaaactg aac 23 <210> 11 <211> twenty one <212> DNA <213> Artificial Sequence <400> 11 accccgaacc actcgctcgc g 21 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <400> 12 tcccaaaata ggtttccacc aacgcc 26 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 tgctttcccc ttgccaacag 20 <210> 14 <211> twenty one <212> DNA <213> Artificial Sequence <400> 14 ggctcctact tgccattcat g 21 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <400> 15 cgctggcgta gcaacagcta ctgga 25 <210> 16 <211> twenty two <212> DNA <213> Artificial Sequence <400> 16 gaagtaggtg tctgttgcac gg 22 <210> 17 <211> twenty four <212> DNA <213> Artificial Sequence <400> 17 gatggccacc ccatcgatga tgcc 24 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <400> 18 cgaactgcac cagacccggr ctcaggt 27 <210> 19 <211> twenty one <212> DNA <213> Artificial Sequence <400> 19 aagttcagta caaagcggga t 21 <210> 20 <211> twenty two <212> DNA <213> Artificial Sequence <400> 20 tgcatggtga atagcaatgg tg 22 <210> twenty one <211> 26 <212> DNA <213> Artificial Sequence <400> twenty one ccaatattgt taaacaagca aagcag 26 <210> twenty two <211> 27 <212> DNA <213> Artificial Sequence <400> twenty two caggactatg aaaaccattt acctaag 27 <210> twenty three <211> 26 <212> DNA <213> Artificial Sequence <400> twenty three aagacacarcctcctggtat agcagt 26 <210> twenty four <211> 30 <212> DNA <213> Artificial Sequence <400> twenty four atggaatcaa tcgsaaaagc tgttcagtca 30 <210> 25 <211> 26 <212> DNA <213> Artificial Sequence <400> 25 tgaaaaggcc cgcraatcat tgctac 26 <210> 26 <211> 34 <212> DNA <213> Artificial Sequence <400> 26 ttgtattcac tcctgactgt attagatcat tagt 34 <210> 27 <211> 31 <212> DNA <213> Artificial Sequence <400> 27 cgaatcattg ctacaagaca taaataatga g 31 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <400> 28 gactgtgaaa caaggggaga ycatgt 26 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <400> 29 gcaaccatac tgataggatg ccttc 25 <210> 30 <211> 31 <212> DNA <213> Artificial Sequence <400> 30 tgctgagcaa tcaaaggagt gcaacatcaa c 31 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <400> 31 cttggaaagtgatgatyatc atttcg 26 <210> 32 <211> 28 <212> DNA <213> Artificial Sequence <400> 32 gtgtaccarc ctgttcttaa aacactga 28 <210> 33 <211> 17 <212> DNA <213> Artificial Sequence <400> 33 ataacaccyc agcacgg 17 <210> 34 <211> twenty one <212> DNA <213> Artificial Sequence <400> 34 caaccgatgc cacatcatta g 21 <210> 35 <211> twenty two <212> DNA <213> Artificial Sequence <400> 35 gccattgctt ccggattaac at 22 <210> 36 <211> 27 <212> DNA <213> Artificial Sequence <400> 36 tacagacaag gataagttgt cttatag 27 <210> 37 <211> twenty one <212> DNA <213> Artificial Sequence <400> 37 cttaaaggaa ttgacggaag g 21 <210> 38 <211> twenty one <212> DNA <213> Artificial Sequence <400> 38 cgggccgggt gaggtttccc g 21 <210> 39 <211> 26 <212> DNA <213> Artificial Sequence <400> 39 caccaccagg agtggagcct gcggct 26 <210> 40 <211> twenty two <212> DNA <213> Artificial Sequence <400> 40 cgtgcagctt gtcacagtgc ag 22 <210> 41 <211> twenty one <212> DNA <213> Artificial Sequence <400> 41 atgggcaacc ctaaggtgaa g 21 <210> 42 <211> 27 <212> DNA <213> Artificial Sequence <400> 42 ctcactcagt gtggcaaagg tgccctt 27 <210> 43 <211> 107 <212> DNA <213> Artificial Sequence <400> 43 agaccaatcc tgtcacctct gactaagggg attttaggat ttgtgttcac gctcaccgtg 60 cccagtgagc gaggactgca gcgtagacgc tttgtccaga atgccct 107 <210> 44 <211> 191 <212> DNA <213> Artificial Sequence <400> 44 agaagacgga ggactaccac aaagcggcag aattgttgtt gattacatgg tgcaaaaacc 60 tgggaaaaca ggaacaattg tctatcaaag gggtgttttg ttgcctcaaa aggtgtggtg 120 cgcgagtggc aggagcaaag tgataaaagg gtcattgcct ttaattggtg aagcagattg 180 ccttcatgaa g 191 <210> 45 <211> 117 <212> DNA <213> Artificial Sequence <400> 45 agcttcacga aggctccaca tacacagcag ctgtccagta caatgttcta gaaaaagatg 60 atgatcccgc atcactaaca atatgggtgc ctatgttcca gtcatctgtg ccagcag 117 <210> 46 <211> 111 <212> DNA <213> Artificial Sequence <400> 46 agtcggacca aaccaaactg aacctccccg cttacggtga ggtgaatggg ttgttgaatc cggcgttggt ggaaacctat tttgggaaca cgcgagcgag tggttcgggg t <210> 47 <211> 91 <212> DNA <213> Artificial Sequence <400> 47 tgctttcccc ttgccaacag acgctggcgt agcaacagct actggaacaa agtctgcgac catcaattat catcatch c <210> 48 <211> 118 <212> DNA <213> Artificial Sequence <400> 48 gatggccacc ccatcgatga tgccccaatg ggcatacatg cacatcgccg gacaggatgc ttcggagtac ctgagtccgg gtctggtgca gttcgcccgt gcaacagaca cctacttc 118 <210> 49 <211> 163 <212> DNA <213> Artificial Sequence <400> 49 aataatcaaa gagacaatca cagaattaat cagacaagaa gtaatatcaa ggaccataaa 60 catacaaagt tcagtacaaa gcgggatccc aatattgtta aacaagcaaa gcagagatct 120 cacacaatta atagagaagt catgcaacag acaggaattg gct 163 <210> 50 <211> 133 <212> DNA <213> Artificial Sequence <400> 50 aagttcagta caaagcggga tcccaatatt gttaaacaag caaagcagag atctcacaca 60 attaatagag aagtcatgca acagacagga attggctcag atatgcgaaa acaccattgc 120 tattcaccat gca 133 <210> 51 <211> 153 <212> DNA <213> Artificial Sequence <400> 51 attccatcaa aagtgaaaag gcccgcaaat cattgctaca agacataaat aatgagttta 60 tggaagttac agaaaagatc caagtggcat cggataatac taatgatcta atacagtcag 120 gagtgaatac aaggcttctt acaattcaga gtc 153 <210> 52 <211> 169 <212> DNA <213> Artificial Sequence <400> 52 acccaaatga gaaggactgt gaaacaaggg gagatcatgt cttttgcgac acagcagcag 60 gaattaatgt tgctgagcaa tcaaaggagt gcaacatcaa catatccact acaaattacc 120 catgcaaagt cagcacagga aggcatccta tcagtatggt tgcactgtc 169 <210> 53 <211> 170 <212> DNA <213> Artificial Sequence <400> 53 tagaaagcgg gacaagtaaa aatgtcttgg aaagtgatga ttatcatttc gttactcata 60 acacctcagc atggactaaa agaaagttat ttagaagaat catgtagtac tataactgaa 120 ggatatctca gtgttttaag aacaggttgg tacaccaatg tctttacatt 170 <210> 54 <211> 115 <212> DNA <213> Artificial Sequence <400> 54 caaccgatgc cacatcatta gctacagaca aggataagtt gtcttatagc attggtgccg 60 atttggggaa gaattttaaa aatcaaggca tagatgttaa tccggaagca atggc 115 <210> 55 <211> 84 <212> DNA <213> Artificial Sequence <400> 55 cttaaaggaa ttgacggaag ggcaccacca ggagtggagc ctgcggctta atttgactca 60 acacgggaaa cctcacccgg cccg 84 <210> 56 <211> 130 <212> DNA <213> Artificial Sequence <400> 56 cgtgcagctt gtcacagtgc agctcactca gtgtggcaaa ggtgcccttg aggttgtcca 60 ggtgagccag gccatcacta aaggcaccga gcactttctt gccatgagcc ttcaccttag 120 ggttgcccat 130

Claims

1. A primer probe set characterized in that, Comprising: 12 groups of primer probes are divided into 4 reaction tubes, reaction tube 1, reaction tube 2, reaction tube 3 and reaction tube 4; The reaction tube 1 comprises: Primer group 1: the nucleotide sequence of the primer group for amplifying influenza A virus is shown as SEQ ID NO: 1 and SEQ ID NO: 2; Probe group 1: the nucleotide sequence of the probe for amplifying influenza A virus is shown as SEQ ID NO: 3; And Primer group 2: the nucleotide sequence of the primer for amplifying influenza B virus is shown as SEQ ID NO: 4 and SEQ ID NO: 5; Probe group 2: the nucleotide sequence of the probe for amplifying influenza B virus is shown as SEQ ID NO: 6; And Primer group 3: the nucleotide sequence of the primer for amplifying respiratory syncytial virus is shown as SEQ ID NO: 7 and SEQ ID NO: 8; Probe group 3: the nucleotide sequence of the probe for amplifying respiratory syncytial virus is shown as SEQ ID NO: 9; And The nucleotide sequence of the primer of human 18s RNA internal standard is shown as SEQ ID NO: 37 and SEQ ID NO: 38, and the nucleotide sequence of the probe of human 18s RNA internal standard is shown as SEQ ID NO: 39; The reaction tube 2 comprises: Primer group 4: the nucleotide sequence of the primer for amplifying Mycoplasma pneumoniae is shown as SEQ ID NO: 10 and SEQ ID NO: 11; Probe group 4: the nucleotide sequence of the probe for amplifying Mycoplasma pneumoniae is shown as SEQ ID NO: 12; And Primer group 6: the nucleotide sequence of the primer for amplifying adenovirus is shown as SEQ ID NO: 16 and SEQ ID NO: 17; Probe group 6: the nucleotide sequence of the probe for amplifying adenovirus is shown as SEQ ID NO: 18; And Primer group 5: the nucleotide sequence of the primer for amplifying Chlamydia pneumoniae is shown as SEQ ID NO: 13 and SEQ ID NO: 14; Probe group 5: the nucleotide sequence of the probe for amplifying Chlamydia pneumoniae is shown as SEQ ID NO: 15; And The nucleotide sequence of the primer of human globin DNA internal standard is shown as SEQ ID NO: 40 and SEQ ID NO: 41, and the nucleotide sequence of the probe of human globin DNA is shown as SEQ ID NO: 42; The reaction tube 3 comprises: Primer group 7: the nucleotide sequence of the primer for amplifying parainfluenza virus 1 is shown as SEQ ID NO: 19 and SEQ ID NO: 20; Probe group 7: the nucleotide sequence of the probe for amplifying parainfluenza virus 1 is shown as SEQ ID NO: 21; And Primer group 8: the nucleotide sequence of the primer for amplifying parainfluenza virus 2 is shown as SEQ ID NO: 22 and SEQ ID NO: 23; Probe group 8: the nucleotide sequence of the probe for amplifying parainfluenza virus 2 is shown as SEQ ID NO: 24; And The nucleotide sequence of the primer set 9 for amplifying parainfluenza virus type 3 is shown as SEQ ID NO: 25 and SEQ ID NO: 26; The nucleotide sequence of the probe set 9 for amplifying parainfluenza virus type 3 is shown as SEQ ID NO: 27; The nucleotide sequence of the primer of the human 18s RNA internal standard is shown as SEQ ID NO: 37 and SEQ ID NO: 38, and the nucleotide sequence of the probe of the human 18s RNA internal standard is shown as SEQ ID NO: 39; The reaction tube 4 comprises: The nucleotide sequence of the primer set 10 for amplifying human metapneumovirus type A is shown as SEQ ID NO: 28 and SEQ ID NO: 29; The nucleotide sequence of the probe set 10 for amplifying human metapneumovirus type A is shown as SEQ ID NO: 30; The nucleotide sequence of the primer set 11 for amplifying human metapneumovirus type B is shown as SEQ ID NO: 31 and SEQ ID NO: 32; The nucleotide sequence of the probe set 11 for amplifying human metapneumovirus type B is shown as SEQ ID NO: 33; The nucleotide sequence of the primer set 12 for amplifying Legionella is shown as SEQ ID NO: 34 and SEQ ID NO: 35; The nucleotide sequence of the probe set 12 for amplifying Legionella is shown as SEQ ID NO: 36; The nucleotide sequence of the primer of the human 18s RNA internal standard is shown as SEQ ID NO: 37 and SEQ ID NO: 38, and the nucleotide sequence of the probe of the human 18s RNA internal standard is shown as SEQ ID NO:

39. The 5' end of the nucleotide sequence is connected with a fluorescent reporter gene, and the 3' end is connected with a quencher gene.

3. The use of the primer probe set of claim 1 in the preparation of a reagent and / or kit for detecting respiratory pathogens.

2. The primer probe set of claim 1, wherein The respiratory pathogens are FluA, FluB, RSV, MP, CP, ADV, HPIV1, HPIV2, HPIV3, HMPV-A, HMPV-B or LP. The primer probe set of claim 1 and an acceptable adjuvant. The reaction solution comprising the primer probe set of claim 1; 4. A test product, characterized in that The reaction solution comprises: Tricine, KOAc, Tween20, glycerol, DMSO, betaine, dNTPs, Tth enzyme, manganese acetate and water.

5. The test product of claim 4, wherein, 6. The use of the primer probe set of claim 1 or the detection product of claim 4 or 5 in the preparation of one or more products for detecting FluA, FluB, RSV, MP, CPn, ADV, HPIV1, HPIV2, HPIV3, HMPV-A, HMPV-B-A, HMPV-A, HMPV-B-B, LP. ​ ​

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