Composition for detecting respiratory tract infection related pathogens

Abstract

The invention belongs to the field of molecular biological detection, and provides a joint detection composition which mainly utilizes a multiple fluorescent PCR analysis method to detect related pathogens, so as to simultaneously realize detection of coronavirus, bordetella pertussis, group A streptococcus, parainfluenza virus, streptococcus pneumoniae, haemophilus influenzae and group B streptococcus in a single-tube reaction system. Or detecting and distinguishing at least four of chlamydia pneumoniae. Meanwhile, the detection sensitivity of the composition is higher and reaches 500 copies / mL, and the detection is more accurate.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to a composition for detecting and distinguishing pathogens related to respiratory tract infection, including coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia pneumoniae. Background Art

[0002] There are at least dozens of known members in the coronavirus family, most of which live in peace with humans. However, seven coronaviruses, such as SARS-CoV and MERS-CoV, which have been produced through multiple mutations, can infect humans.

[0003] Chlamydia pneumoniae (CP) infection is a respiratory infectious disease. The transmission route is through the droplets or sneezes of patients with Chlamydia pneumoniae, which are inhaled into the respiratory tract by healthy people; Chlamydia pneumoniae grows, reproduces and invades the latter's respiratory cells, thus causing infection.

[0004] Streptococcus pneumoniae (SP) is a bacterium. It is a diplococcus with short double-row chains of Gram chromosomes. Generally, pneumococci can cause patients to suffer from pneumonia, meningitis, bronchitis and other diseases. The transmission route is through the droplets or sneezes of pneumococcal patients, which are inhaled into the respiratory tract by healthy people; the onset is often acute (a few children first have mild upper respiratory tract infection symptoms), and the symptoms of poisoning are severe. Sudden high fever, body temperature can reach 40℃~41℃, headache, chest pain, shortness of breath, irritability, often no cough or light cough in the early stage, and sick children often do not spit. Older children may have chills and spit up rust-colored sputum, but some children still cough up yellow purulent sputum. Severely ill children may have toxic encephalopathy manifestations such as convulsions, delirium and coma.

[0005] Haemophilus influenzae (HI) is a naturally occurring bacterium that occurs only in humans. In infants and children, H. influenzae type b causes bacteremia and acute bacterial meningitis. Occasionally, it causes cellulitis, osteomyelitis, and joint infections.

[0006] The main diseases that parainfluenza viruses can cause include the common cold, bronchitis, bronchiolitis and pneumonia. Rhinoviruses, coronaviruses, adenoviruses, etc. that are similar to parainfluenza viruses can also cause the common cold and even bronchitis. Parainfluenza viruses are divided into four types, namely PIV1-4.

[0007] A single bacterium of group A streptococcus is spherical or oval, with a diameter of about 1 μm. It is arranged in chains or pairs, and the length of the chains varies depending on environmental factors. Gram staining is positive. As the culture time increases or the bacteria die, it may become Gram staining negative. There are no flagella and spores are not formed. Group A streptococci have hair-like M proteins and pilus structures wrapped with lipoteichoic acid. Most group A streptococci can form hyaluronic acid capsules in the early stage of culture (2 to 4 hours). As the culture time increases, it is degraded and disappears due to the hyaluronidase produced by the bacteria.

[0008] Therefore, there is a need in the art for a product that can simply and quickly detect the above-mentioned pathogens with high sensitivity and good specificity, which is conducive to providing timely clinical treatment strategies. Summary of the invention

[0009] In view of this, in a first aspect, the present invention provides a composition for detecting pathogens related to respiratory tract infection, comprising upstream and downstream primers and probes for detecting at least 4 of the following related pathogens:

[0010] Upstream and downstream primers and probes for detecting coronavirus as shown in SEQ ID NOs: 1 to 3;

[0011] Upstream and downstream primers and probes for detecting Bordetella pertussis as shown in SEQ ID NOs: 4 to 6;

[0012] Upstream and downstream primers and probes for detecting Group A Streptococcus as shown in SEQ ID NOs: 7 to 9;

[0013] Upstream and downstream primers and probes for detecting parainfluenza virus as shown in SEQ ID NOs: 10 to 12;

[0014] Upstream and downstream primers and probes for detecting Streptococcus pneumoniae as shown in SEQ ID NOs: 13 to 15;

[0015] Upstream and downstream primers and probes for detecting Haemophilus influenzae as shown in SEQ ID NOs: 16 to 18; or

[0016] The upstream and downstream primers and probes for detecting Chlamydia pneumoniae are shown in SEQ ID NOs: 19-21.

[0017] The joint detection composition provided by the present invention mainly uses a multiplex fluorescent PCR analysis method to detect relevant pathogens, thereby simultaneously realizing the detection and differentiation of at least four pathogens including coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia pneumoniae in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. At the same time, the composition of the present invention has a higher detection sensitivity of 500 copies / mL, and its detection is more accurate than the melting curve, which is conducive to providing prevention policies in a timely manner.

[0018] Furthermore, the composition includes upstream and downstream primers and probes of at least 5 of the above pathogens.

[0019] Furthermore, the composition includes upstream and downstream primers and probes of at least 6 of the above pathogens.

[0020] The present invention provides a composition for detecting pathogens related to respiratory tract infection, comprising:

[0021] Upstream and downstream primers and probes for detecting coronavirus as shown in SEQ ID NOs: 1 to 3;

[0022] Upstream and downstream primers and probes for detecting Bordetella pertussis as shown in SEQ ID NOs: 4 to 6;

[0023] Upstream and downstream primers and probes for detecting Group A Streptococcus as shown in SEQ ID NOs: 7 to 9;

[0024] Upstream and downstream primers and probes for detecting parainfluenza virus as shown in SEQ ID NOs: 10 to 12;

[0025] Upstream and downstream primers and probes for detecting Streptococcus pneumoniae as shown in SEQ ID NOs: 13 to 15;

[0026] Upstream and downstream primers and probes for detecting Haemophilus influenzae as shown in SEQ ID NOs: 16 to 18; and

[0027] The upstream and downstream primers and probes for detecting Chlamydia pneumoniae are shown in SEQ ID NOs: 19-21.

[0028] Furthermore, the composition includes upstream and downstream primers and probes for detecting the internal standard.

[0029] In some specific embodiments, the internal standard is a human internal standard gene.

[0030] In a specific embodiment, the internal standard is a human housekeeping gene.

[0031] In a specific embodiment, the composition includes upstream and downstream primers and probes for detecting internal standards as shown in SEQ ID NOs: 22-24.

[0032] Furthermore, the fluorescent groups of different probes in the composition of the present invention are different from each other and do not interfere with each other.

[0033] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX, CY5, CY7 and CY5.5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0034] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe sets. In the present invention, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0035] The composition of the present invention can be combined into any combination of the corresponding eight targets. Those skilled in the art can combine the corresponding targets as needed, that is, combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0036] For example, it may include any 7 of the above 8 groups of primers and probes, it may include any 6 of the above 8 groups of primers and probes, it may include any 5 of the above 8 groups of primers and probes, it may include any 4 of the above 8 groups of primers and probes, it may include any 3 of the above 8 groups of primers and probes, it may include any 2 of the above 6 groups of primers and probes, and it may also include any 1 of the above 8 groups of primers and probes.

[0037] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0038] In a specific embodiment, the fluorescent reporter group of the coronavirus probe is FAM; the fluorescent reporter group of the Bordetella pertussis probe is HEX; the fluorescent reporter group of the group A Streptococcus probe is ROX; the fluorescent reporter group of the parainfluenza virus probe is CY5; the fluorescent reporter group of the Streptococcus pneumoniae probe is Quasar 705; the fluorescent reporter group of the Haemophilus influenzae probe is ATTO425; and the fluorescent reporter group of the Chlamydia pneumoniae probe is CY7.

[0039] In a specific embodiment, the fluorescent reporter group of the internal standard is AF 405.

[0040] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0041] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, Dabcyl BHQ-0, BHQ1, BHQ2 or BHQ3.

[0042] Furthermore, the dosage of a single primer in the composition is 0.01 to 0.5 μM; the dosage of a single probe in the composition is 0.005 to 0.2 μM.

[0043] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0044] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0045] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0046] In a second aspect, the present invention provides the use of the above-mentioned composition of the present invention in the preparation of a kit for detecting pathogens related to respiratory tract infections, wherein the type of the related pathogens is one or more of coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydia pneumoniae.

[0047] In a third aspect, the present invention provides a kit for detecting pathogens associated with respiratory tract infections, the kit comprising the composition of the present invention as described above.

[0048] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0049] In a specific embodiment, the negative control is at least one of DEPC H2O, saline, and an internal standard gene. The positive control is at least one of a pseudovirus, plasmid, or fragment gene of coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydia pneumoniae.

[0050] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0051] Furthermore, the amplification reagent includes dNTP(U)s, PCR buffer, DNA polymerase, and Mg 2+ At least one of .

[0052] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0053] Furthermore, the concentration of the DNA polymerase is 1 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0054] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, Mg 2+ , dNTP(U)s, primers, probes, UNG enzyme and PCR buffer.

[0055] PCR buffer is composed of Tris-HCl, MgCl2, KCl, Triton X-100, etc. The total volume in a single PCR reaction tube is generally 20 μl to 200 μl.

[0056] In a fourth aspect, a method for detecting pathogens associated with respiratory tract infection is provided, the method comprising the following steps:

[0057] 1) Extracting nucleic acid from the sample to be tested;

[0058] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0059] 3) Obtain and analyze the results.

[0060] In the present invention, the sample used for detection can be serum, blood, etc., but is not limited thereto.

[0061] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0062] Reverse transcription, temperature is 50-65°C, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99°C, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95°C, time is 5-30 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0063] In a specific embodiment, a method for detecting pathogens associated with respiratory tract infection for non-diagnostic purposes is provided, the method comprising the following steps:

[0064] 1) Extracting nucleic acid from the sample to be tested;

[0065] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0066] 3) Obtain and analyze the results.

[0067] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0068] Reverse transcription, temperature is 50-65°C, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99°C, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95°C, time is 5-30 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0069] In a specific embodiment, a composition is provided for use in preparing a reagent for detecting pathogens associated with respiratory tract infections, wherein the use detection comprises the following steps:

[0070] 1) Extracting nucleic acid from the sample to be tested;

[0071] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0072] 3) Obtain and analyze the results.

[0073] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0074] Reverse transcription, temperature is 50-65°C, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99°C, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95°C, time is 5-30 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0075] In this context, the term "non-diagnostic purpose" means that the method is not intended to obtain information on whether an individual is infected with the above-mentioned pathogens and suffers from the infection. For example, the method can detect whether the environment and culture have the above-mentioned pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figures 1 to 4 A graph showing the test results of different sample combinations of the composition of the present invention;

[0077] Figures 5 to 11 The sensitivity results of the composition of the present invention are shown in Figure 1 (coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia pneumoniae);

[0078] Fig.12 It is a specific result diagram of the composition of the present invention;

[0079] Figures 13 to 19 The anti-interference results of the composition of the present invention are shown in Figure 1 (coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, and Chlamydia pneumoniae);

[0080] Fig. 20 It is a test result diagram of the comparative example composition of the present invention. DETAILED DESCRIPTION

[0081] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0082] Example 1: Primers and probes used in the present invention

[0083] Table 1

[0084]

[0085] Among them, the fluorescent reporter group of the coronavirus probe is FAM; the fluorescent reporter group of the Bordetella pertussis probe is HEX; the fluorescent reporter group of the group A Streptococcus probe is ROX; the fluorescent reporter group of the parainfluenza virus probe is CY5; the fluorescent reporter group of the Streptococcus pneumoniae probe is Quasar 705; the fluorescent reporter group of the Haemophilus influenzae probe is ATTO425; the fluorescent reporter group of the Chlamydia pneumoniae probe is CY7; and the fluorescent reporter group of the internal standard probe is AF 405.

[0086] Example 2: Method for detecting pathogens associated with respiratory tract infection

[0087] 1. Reagent preparation

[0088] Take out each component in the box, place it at room temperature, wait for the temperature to equilibrate to room temperature, mix well and centrifuge for a short time before use.

[0089] According to the number of samples to be tested, negative controls, and positive controls, take the corresponding amount of components in proportion (26 μL / person of PCR reaction solution + 4 μL / person of enzyme mixture), mix them thoroughly to form a PCR mixture, and centrifuge it for standby use.

[0090] Transfer the above prepared reagents to the sample processing area for later use.

[0091] 2. Sample processing and sample addition

[0092] The test sample of the present invention is a throat swab / sputum. The magnetic bead method is used to extract viral nucleic acid, and the following operations are performed in the sample processing room:

[0093] Take a 1.5mL cryotube and add glass beads. Add 300uL of the treated sample and 300uLTE to the 1.5mL cryotube containing mixed glass beads and grind using a grinder. When the grinding is completed, take out the cryotube and centrifuge it instantly. Take out the supernatant liquid as the sample to be tested for later use.

[0094] Take a number of 1.5mL centrifuge tubes according to the number of samples to be tested, and add 300μL of the sample to be tested to each tube;

[0095] Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cover the tube, shake and mix for 30 seconds, and heat at 60°C for 10 minutes.

[0096] Let stand at room temperature for 1 min, centrifuge at low speed, place the centrifuge tube on the magnetic separator, and slowly aspirate and discard the waste liquid after 5 min (*Be careful not to touch the magnetic beads adsorbed on the inner side of the tube wall);

[0097] Add 200 μL of washing solution 1 and 600 μL of washing solution 2, shake and mix for 30 seconds, centrifuge at low speed, and place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, completely aspirate and discard the liquid.

[0098] Place the centrifuge tube in a centrifuge and centrifuge it at low speed for a short time, then place the centrifuge tube in a magnetic separator again. Magnetic suction for 3 minutes to completely absorb the liquid at the bottom of the tube.

[0099] Add 30-100 μL of elution buffer S (80 μL is recommended), shake and mix for 30 seconds to elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, and let it stand at room temperature for 3 minutes; centrifuge at a low speed, place the centrifuge tube on the magnetic separator again for 3 minutes, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0100] Pipette 20 μL of the above-treated samples, positive controls and negative controls and add them to the corresponding 0.2 mL PCR reaction tubes, add 2 and 30 μL of PCR mixture to each tube, and cover the tubes.

[0101] The sample detected by the present invention is sputum. The real-time fluorescence PCR reaction solution is configured according to the following Table 2:

[0102] Table 2

[0103]

[0104] PCR amplification:

[0105] On the SLAN-48S fully automatic medical PCR analysis system, PCR amplification is performed according to a certain temperature and time setting program. The PCR amplification program is set as shown in Table 3 below:

[0106] Table 3

[0107]

[0108] For samples with a typical S-shaped amplification curve detected in the FAM channel and Ct≤40, they are reported as Cov positive; for samples with no typical S-shaped amplification curve detected in the FAM channel, or Ct>40, and an amplification curve in the AF 405 channel and Ct≤40, they are reported as Cov negative.

[0109] For samples with a typical S-shaped amplification curve detected in the HEX channel and Ct≤40, they are reported as BP positive; for samples with no typical S-shaped amplification curve detected in the HEX channel, or Ct>40, and an amplification curve in the AF 405 channel and Ct≤40, they are reported as BP negative.

[0110] For samples with a typical S-shaped amplification curve detected in the ROX channel and Ct≤40, they are reported as GAS positive; for samples with no typical S-shaped amplification curve detected in the ROX channel, or Ct>40, and an amplification curve in the AF 405 channel and Ct≤40, they are reported as GAS negative.

[0111] For samples with a typical S-shaped amplification curve detected in the CY5 channel and Ct≤40, they are reported as PIVs positive; for samples with no typical S-shaped amplification curve detected in the CY5 channel, or Ct>40, and an amplification curve in the AF 405 channel and Ct≤40, they are reported as PIVs negative.

[0112] For samples with a typical S-shaped amplification curve detected by the QUASAR 705 channel and Ct≤40, they are reported as SP positive; for samples with no typical S-shaped amplification curve detected by the QUASAR 705 channel, or Ct>40, and an amplification curve with Ct≤40 on the AF 405 channel, they are reported as SP negative.

[0113] For samples with a typical S-shaped amplification curve detected in the ATTO 425 channel and Ct≤40, it is reported as HI positive; for samples with no typical S-shaped amplification curve detected in the ATTO 425 channel, or Ct>40, and an amplification curve in the AF 405 channel and Ct≤40, it is reported as HI negative.

[0114] For samples with a typical S-type amplification curve detected in the CY7 channel and Ct≤40, it is reported as CP positive; for samples with no typical S-type amplification curve detected in the CY7 channel, or Ct>40, and an amplification curve in the AF 405 channel and Ct≤40, it is reported as CP negative. The details are shown in Table 4.

[0115] Table 4. Interpretation of test results

[0116]

[0117]

[0118] Example 3: Test results of the test samples of the composition of the present invention

[0119] The primers and probes shown in Example 1 were verified according to the method of Example 2 on samples of coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, and Haemophilus influenzae. The results showed that the composition of the present invention can detect and distinguish any combination of the above-mentioned related pathogens. For example, the composition of the present invention detects:

[0120] Coronavirus, parainfluenza virus, group A streptococcus, pneumococcus ( Figure 1 );

[0121] Coronavirus, Bordetella pertussis, Group A Streptococcus, Parainfluenza virus, Haemophilus influenzae ( Figure 2 );

[0122] Coronavirus, Bordetella pertussis, Group A Streptococcus, Parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae ( Figure 3 );

[0123] Coronavirus, Bordetella pertussis, Group A Streptococcus, Parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae ( Figure 4 ).

[0124] These figures prove that the present invention is a 7-plex PCR that can amplify the above-mentioned 7 targets at one time and can be distinguished using an 8-channel PCR instrument. It also proves that any corresponding pair combination of the composition of the present invention can detect any corresponding combination of pathogens.

[0125] Example 4: Sensitivity of the composition of the present invention

[0126] Using the composition of Example 1 of the present invention, LOD (sensitivity) detection was performed on samples of 5000, 2500, 1000, 500, and 50 copies / ml to simulate clinical samples, and multiplex PCR detection was performed on a fluorescent quantitative PCR instrument. The results are as follows: Figures 5 to 11 As shown, it is shown that each channel can still accurately detect samples as low as 500 copies / mL, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0127] Example 5. Specificity of the composition of the present invention

[0128] The composition of the present invention has no cross reaction with common pathogens and other pathogens with similar infection symptoms (Escherichia coli, Streptococcus pyogenes, Acinetobacter baumannii, Pneumocystis jiroveci, Klebsiella pneumoniae, Pseudomonas aeruginosa, Legionella pneumophila, Staphylococcus aureus, Epstein-Barr virus, human metapneumovirus, measles virus, mumps virus, norovirus, rotavirus, Candida albicans, Mycobacterium tuberculosis). The results are as follows Fig.12 As shown, it is shown that the composition of the present invention has good specificity.

[0129] Example 6: Anti-interference performance of the composition of the present invention

[0130] The composition of the present invention is not affected by 50 μg / mL dexamethasone, 50 μg / mL cefmenoxime hydrochloride, 100 μg / mL ribavirin, 100 μg / mL azithromycin, 320 μg / mL budesonide, 125 μg / mL phenylephrine, 50 μg / mL beclomethasone, 100 μg / mL mometasone, 200 μg / mL fluticasone, 200 μg / mL histamine hydrochloride, 100 μg / mL lopinavir, 100 μg / mL triamcinolone acetonide, 100 μg / mL arbidol, and 10 μg / mL heme, respectively. The results are as follows: Figures 13 to 19 shown.

[0131] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0132] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be ruled out at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. In order to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0133] Therefore, the inventors also designed some other primers and probes to form a different detection system 2 (sequence not shown), which is also used to detect the above pathogens. Fig. 20 As shown in the figure, it can be seen that only part of the amplification curve appears, the amplification curve has a low increase and poor repeatability, and missed detection occurs.

Claims

1. A composition for detecting pathogens related to respiratory tract infection, comprising upstream and downstream primers and probes for detecting at least 4 of the following related pathogens: Includes upstream and downstream primers and probes for detecting at least 4 of the following related pathogens: Upstream and downstream primers and probes for detecting coronavirus as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Bordetella pertussis as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Group A Streptococcus as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting parainfluenza virus as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Streptococcus pneumoniae as shown in SEQ ID NOs: 13 to 15; Upstream and downstream primers and probes for detecting Haemophilus influenzae as shown in SEQ ID NOs: 16 to 18; or The upstream and downstream primers and probes for detecting Chlamydia pneumoniae are shown in SEQ ID NOs: 19-21.

2. The composition according to claim 1, characterized in that The composition comprises: Upstream and downstream primers and probes for detecting coronavirus as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Bordetella pertussis as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Group A Streptococcus as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting parainfluenza virus as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Streptococcus pneumoniae as shown in SEQ ID NOs: 13 to 15; Upstream and downstream primers and probes for detecting Haemophilus influenzae as shown in SEQ ID NOs: 16 to 18; and The upstream and downstream primers and probes for detecting Chlamydia pneumoniae are shown in SEQ ID NOs: 19-21.

3. The composition according to claim 1, characterized in that The composition further comprises: The upstream and downstream primers and probes for detecting the internal standard are shown in SEQ ID NOs: 22-24.

4. The composition according to claim 1, characterized in that The fluorescent groups of different probes in the composition are different from each other and do not interfere with each other.

5. The composition according to claim 4, characterized in that The fluorescent reporter group of the coronavirus probe is FAM; the fluorescent reporter group of the Bordetella pertussis probe is HEX; the fluorescent reporter group of the group A Streptococcus probe is ROX; the fluorescent reporter group of the parainfluenza virus probe is CY5; the fluorescent reporter group of the Streptococcus pneumoniae probe is Quasar 705; the fluorescent reporter group of the Haemophilus influenzae probe is ATTO425; the fluorescent reporter group of the Chlamydia pneumoniae probe is CY7; and the fluorescent reporter group of the internal standard probe is AF 405.

6. The composition according to any one of claims 1 to 5, characterized in that The components of the composition are present in admixture.

7. Use of the composition according to any one of claims 1 to 6 in preparing a kit for detecting pathogens related to respiratory tract infection, wherein: The type of the relevant pathogen is one or more of coronavirus, Bordetella pertussis, Group A Streptococcus, parainfluenza virus, Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydia pneumoniae.

8. A kit for detecting pathogens associated with respiratory tract infection, the kit comprising the composition according to any one of claims 1 to 6.

9. The kit according to claim 8, characterized in that The kit also includes at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a nucleic acid amplifying reagent.

10. Use of a composition for preparing a reagent for detecting pathogens associated with respiratory tract infection, the detection comprising the following steps: 1) Extracting nucleic acid from the sample to be tested; 2) performing fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 6 or the kit according to claim 8 or 9; 3) Obtain and analyze the results.

Images