Primer probe combination for broad spectrum screening of common respiratory pathogens and application thereof

Abstract

This invention relates to the field of biomedical technology, and more particularly to primer-probe combinations for broad-spectrum screening of common respiratory pathogens and their applications. Through screening and optimization, this invention has obtained a primer-probe combination for nucleic acid detection of respiratory pathogens for broad-spectrum screening of common acute respiratory infections and community-acquired pneumonia. The primer-probe combination described in this invention covers 18 respiratory pathogens and possesses advantages such as high accuracy, good specificity, strong anti-interference ability, good inclusiveness, high sensitivity, and superconductivity. Combined with fully enclosed single-tube PCR amplification, it can achieve parallel detection of 20 targets, greatly improving the early diagnosis of respiratory diseases with complex pathogen structures, and is of great significance for the prevention and treatment of infectious diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to primer-probe combinations for broad-spectrum screening of common respiratory pathogens and their applications. Background Technology

[0002] Respiratory tract infections are among the most common diseases worldwide. They are divided into upper and lower respiratory tract infections. Upper respiratory tract infections are often accompanied by symptoms such as rhinitis, cough, pharyngitis, and fever, while lower respiratory tract infections include tracheitis, bronchitis, and pneumonia. Acute respiratory tract infection (ARTI) can occur in any part of the respiratory tract and is one of the most common infectious diseases, especially lower respiratory tract infections, which are a major cause of all infectious diseases. Many pathogens can cause respiratory tract infections, including bacteria, viruses, mycoplasma, and chlamydia. Studies show that mycoplasma pneumoniae pneumonia accounts for a high percentage of community-acquired pneumonia (CAP). In domestic clinical diagnosis, due to limitations in treatment conditions, many patients with respiratory tract infections cannot receive a more accurate diagnosis and are often treated with broad-spectrum antibiotics. Clinically, approximately 25%–20% of antibiotic use is inappropriate. This not only fails to achieve optimal treatment results but may also delay treatment, induce bacterial resistance, and lead to more complex superinfections. Respiratory infections often involve mixed infections of multiple pathogens. Clinicians need to consider which pathogen or the combined effect of multiple pathogens is the main reason for hospitalization of pneumonia patients.

[0003] Due to the complex pathogen spectrum and high incidence of respiratory diseases, there are very few multi-sample nucleic acid testing products available. The specificity and sensitivity of diagnostic reagents for the early diagnosis of ARTIs and CAP (Acute Respiratory Disease) in respiratory diseases still need improvement. Furthermore, most ARTIs and CAPs are acute illnesses characterized by concentrated outbreaks, thus requiring testing methods that are easy to operate, rapid, and have high throughput. Therefore, developing multi-sample testing products or methods with high specificity, high sensitivity, ease of operation, rapidity, and high throughput is particularly necessary. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a primer-probe combination for broad-spectrum screening of common respiratory pathogens and its application.

[0005] This invention provides a primer-probe combination for detecting respiratory pathogens, wherein the target fragment of the primer-probe combination includes:

[0006] Nucleotide sequences such as the influenza B virus (IFB) target fragment shown in SEQ ID NO.82; and / or

[0007] Nucleotide sequences such as the Mycoplasma pneumoniae (MP) target fragment shown in SEQ ID NO.83; and / or

[0008] The ORF1ab gene target fragment of the novel coronavirus (SARS-CoV-2) with a nucleotide sequence as shown in SEQ ID NO.84; and / or

[0009] Nucleotide sequences such as the parainfluenza virus type 1 (PIV-1) target fragment shown in SEQ ID NO. 85; and / or

[0010] Nucleotide sequences such as the parainfluenza virus type 2 (PIV2) target fragment shown in SEQ ID NO. 86; and / or

[0011] Nucleotide sequences such as the parainfluenza virus type 3 (PIV3) target fragment shown in SEQ ID NO. 87; and / or

[0012] Nucleotide sequences such as the parainfluenza virus type 4 (PIV4) target fragment shown in SEQ ID NO. 88; and / or

[0013] Nucleotide sequences of coronavirus 229E (HCOV-229E) target fragments as shown in SEQ ID NO.89; and / or

[0014] Nucleotide sequences such as the coronavirus HKU(HCOV-HKU1)1 target fragment shown in SEQ ID NO.90; and / or

[0015] Nucleotide sequences of coronavirus OC43 (HCOV-OC43) target fragments as shown in SEQ ID NO.91; and / or

[0016] Nucleotide sequences of coronavirus NL63 (HCOV-NL63) target fragments as shown in SEQ ID NO.92; and / or

[0017] Nucleotide sequences such as the bocavirus (HBOV) target fragment shown in SEQ ID NO.93; and / or

[0018] Nucleotide sequences such as the Chlamydia pneumoniae (CP) target fragment shown in SEQ ID NO.94; and / or

[0019] Nucleotide sequences such as the rhinovirus (HRV) target fragment shown in SEQ ID NO. 95; and / or

[0020] Nucleotide sequences such as the HMPV target fragment shown in SEQ ID NO.96; and / or

[0021] Nucleotide sequences such as the adenovirus (HADV) target fragment shown in SEQ ID NO.97; and / or

[0022] Nucleotide sequences such as the respiratory syncytial virus (RSV) target fragment shown in SEQ ID NO. 98; and / or

[0023] Nucleotide sequences of the N gene target fragment of the novel coronavirus (SARS-CoV-2) as shown in SEQ ID NO.99; and / or

[0024] Nucleotide sequences such as SEQ ID NO.100 are target fragments of influenza A virus (IFA).

[0025] Furthermore, the target fragment also includes an internal standard target fragment with a nucleotide sequence as shown in SEQ ID NO.101.

[0026] The target fragments (or primer-probe combinations) for detecting respiratory pathogens described in this invention are target fragments (or primer-probe combinations) for detecting 18 respiratory pathogens (19 target sequences). Specifically, the 18 respiratory pathogens (19 target sequences) include: influenza B virus (IFB), mycoplasma pneumoniae (MP), novel coronavirus (SARS-CoV-2, ORF1ab gene target fragment and N gene target fragment), parainfluenza virus type 1 (PIV-1), and parainfluenza virus type 2 (PIV-2). The following viruses are listed: V2, parainfluenza virus type 3 (PIV3), parainfluenza virus type 4 (PIV4), coronavirus 229E (HCOV-229E), coronavirus HKU (HCOV-HKU1), coronavirus OC43 (HCOV-OC43), coronavirus NL63 (HCOV-NL63), bocavirus (HBOV), chlamydia pneumoniae (CP), rhinovirus (HRV), metapneumovirus (HMPV), adenovirus (HADV), respiratory syncytial virus (RSV), and influenza A virus (IFA).

[0027] In the specific detection of the above 18 respiratory pathogens, the pathogens can be combined for detection according to actual needs, which can generate combinations of target sequences (or primer-probe combinations) containing different pathogens. Each combination requires an internal standard as a reference. Specifically, the combination of target fragments includes at least one of the following combinations 1 to 3:

[0028] Combination 1: A combination containing any one of the target fragments from the 18 respiratory pathogens and an internal control target fragment;

[0029] Combination 2: A combination containing any number of target fragments and internal control target fragments from the 18 respiratory pathogens;

[0030] Combination 3: A combination containing all target fragments and internal control target fragments from the aforementioned 18 respiratory pathogens;

[0031] In specific embodiments of the present invention, the target fragments of the aforementioned pathogens can be arbitrarily and randomly combined, and the detection results are not limited by the types of pathogens. Regardless of the combination, they exhibit high accuracy, precision, sensitivity, and stability. When the equipment allows, the detection of 18 pathogens simultaneously yields better results and saves detection time.

[0032] Furthermore, in this invention, the primer set and probe for detecting influenza B virus (IFB) include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2, and a probe with a nucleotide sequence as shown in SEQ ID NO.41 and / or SEQ ID NO.42; and / or

[0033] A primer set and probe for the detection of Mycoplasma pneumoniae (MP), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 3, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 4, and a probe with a nucleotide sequence as shown in SEQ ID NO. 43 and / or SEQ ID NO. 44; and / or

[0034] Primer sets and probes for the detection of the novel coronavirus (SARS-CoV-2), comprising: primer set and probe combination 1 and primer set and probe combination 2; primer set and probe combination 1 comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 5, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 6, and a probe with a nucleotide sequence as shown in SEQ ID NO. 45 and / or SEQ ID NO. 46; primer set and probe combination 2 comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 35, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 36, and a probe with a nucleotide sequence as shown in SEQ ID NO. 75 and / or SEQ ID NO. 76; and / or

[0035] A primer set and probe for detecting parainfluenza virus type 1 (PIV-1), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 8, and a probe with a nucleotide sequence as shown in SEQ ID NO. 47 and / or SEQ ID NO. 48; and / or

[0036] A primer set and probe for detecting parainfluenza virus type 2 (PIV2), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 9, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 10, and a probe with a nucleotide sequence as shown in SEQ ID NO. 49 and / or SEQ ID NO. 50; and / or

[0037] A primer set and probe for detecting parainfluenza virus type 3 (PIV3), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 11, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 12, and a probe with a nucleotide sequence as shown in SEQ ID NO. 51 and / or SEQ ID NO. 52; and / or

[0038] A primer set and probe for detecting parainfluenza virus type 4 (PIV4), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 13, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 14, and a probe with a nucleotide sequence as shown in SEQ ID NO. 53 and / or SEQ ID NO. 54; and / or

[0039] A primer set and probe for the detection of coronavirus 229E (HCOV-229E), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 15, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 16, and a probe with a nucleotide sequence as shown in SEQ ID NO. 55 and / or SEQ ID NO. 56; and / or

[0040] A primer set and probe for the detection of coronavirus HKU (HCOV-HKU1), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 17, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 18, and a probe with a nucleotide sequence as shown in SEQ ID NO. 57 and / or SEQ ID NO. 58; and / or

[0041] A primer set and probe for the detection of coronavirus OC43 (HCOV-OC43), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 19, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 20, and a probe with a nucleotide sequence as shown in SEQ ID NO. 59 and / or SEQ ID NO. 60; and / or

[0042] A primer set and probe for the detection of coronavirus NL63 (HCOV-NL63), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 21, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 22, and a probe with a nucleotide sequence as shown in SEQ ID NO. 61 and / or SEQ ID NO. 62; and / or

[0043] A primer set and probe for the detection of boca virus (HBOV), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 23, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 24, and a probe with a nucleotide sequence as shown in SEQ ID NO. 63 and / or SEQ ID NO. 64; and / or

[0044] A primer set and probe for the detection of Chlamydia pneumoniae (CP), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 25, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 26, and a probe with a nucleotide sequence as shown in SEQ ID NO. 65 and / or SEQ ID NO. 66; and / or

[0045] A primer set and probe for rhinovirus (HRV) detection, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 27, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 28, and a probe with a nucleotide sequence as shown in SEQ ID NO. 67 and / or SEQ ID NO. 68; and / or

[0046] A primer set and probe for the detection of HMPV, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 29, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 30, and a probe with a nucleotide sequence as shown in SEQ ID NO. 69 and / or SEQ ID NO. 70; and / or

[0047] A primer set and probe for adenovirus (HADV) detection, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 31, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 32, and a probe with a nucleotide sequence as shown in SEQ ID NO. 71 and / or SEQ ID NO. 72; and / or

[0048] A primer set and probe for the detection of respiratory syncytial virus (RSV), comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 33, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 34, and a probe with a nucleotide sequence as shown in SEQ ID NO. 73 and / or SEQ ID NO. 74; and / or

[0049] Primer set and probe for the detection of influenza A virus (IFA), the primer set and probe comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 37, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 38, and a probe with a nucleotide sequence as shown in SEQ ID NO. 77 and / or SEQ ID NO. 78.

[0050] In this invention, two probes are used for each specific pathogen, and the nucleotide sequences of the two probes are partially inversely complementary.

[0051] In this invention, two probes are provided for each specific pathogen. Each of the two probes simultaneously possesses a fluorescent reporter group and a fluorescent quencher group. The fluorescent reporter group includes FAM, HEX, JOE, TET, VIC, ROX, CY3, CY5, CY7, or NED, with FAM, VIC, ROX, CY3, CY5, TET, or JOE being more preferred. The quencher group includes BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, BBQ 650, or TAMRA, with BHQ1, BHQ2, BHQ3, or Dabcyl being more preferred. The other probe has a blocking group, which includes phosphte, dNTP, NH2, or O-ME, with phosphte or dNTP being more preferred.

[0052] Furthermore, the primer-probe combination described in this invention also includes a primer set and a probe for detecting an internal reference.

[0053] The primer set and probes used for detecting the internal reference include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 39, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 40, and probes with nucleotide sequences as shown in SEQ ID NO. 79, SEQ ID NO. 80, and / or SEQ ID NO. 81.

[0054] Correspondingly, the primer-probe combination can also be combined in the form of target fragment combinations as shown in Combinations 1 to 3 to obtain different groups of the above-mentioned preferred primer-probe combinations for simultaneous detection of different pathogens.

[0055] The present invention provides reagents comprising the primer-probe combination described herein, and further comprising solvents, stabilizers, preservatives and / or antioxidants, etc., which are not limited thereto.

[0056] The present invention provides a kit comprising at least one of the primer-probe combination or reagents described in the present invention and PCR reagents.

[0057] Furthermore, the PCR detection reagent includes DNA polymerase, reverse transcriptase, RNase inhibitors, and / or dNTPs.

[0058] The DNA polymerase includes Taq polymerase.

[0059] Furthermore, the kit also includes a negative control and a positive control; the negative control is a human internal standard gene; the positive control is a target amplified sequence containing the detection of IFB, RSV, CP and the human internal standard gene.

[0060] This invention provides the application of at least one of the following (i) to (iii) in the detection of pathogenic respiratory samples for non-diagnostic purposes:

[0061] i) The primer-probe combination described in this invention;

[0062] ii) The reagents described in this invention;

[0063] iii) The reagent kit described in this invention.

[0064] Furthermore, the respiratory sample includes sputum, saliva, and / or swabs.

[0065] This invention provides a method for detecting respiratory pathogens for non-diagnostic purposes, which involves detecting a sample using at least one of the following methods (i) to (iii):

[0066] i) The primer-probe combination described in this invention;

[0067] ii) The reagents described in this invention;

[0068] iii) The reagent kit described in this invention.

[0069] In this invention, the method for detecting respiratory pathogens for non-diagnostic purposes is a method for detecting multiple respiratory pathogens for non-diagnostic purposes; however, in specific use, it can also be used for the detection of single pathogens, and this invention does not limit it.

[0070] Furthermore, the detection method includes: mixing the sample, the reagent (primer-probe mixture) of the present invention, and the PCR reagent (enzyme mixture) in the reagent of the present invention, and then performing PCR detection;

[0071] In this invention, the volume ratio of the sample, the reagent (primer-probe mixture) described in this invention, and the PCR reagent (enzyme mixture) in the kit is 5:3:4;

[0072] During the reaction, in the primer-probe combination, the concentration of the upstream primer is 100nM to 300nM, preferably 200nM; the concentration of the downstream primer is 100nM to 300nM, preferably 200nM; and the concentration of the probe is 50nM to 200nM, preferably 120nM.

[0073] During the reaction, the DNA polymerase in the PCR amplification reagent is Taq polymerase; the concentration of Taq polymerase is 1U / 20μL to 3U / 20μL, preferably 2U; the concentration of reverse transcriptase is 1U / 20μL to 4U / 20μL, preferably 2U; the concentration of RNase inhibitor is 5U / 20μL to 20U / 20μL, preferably 10U; and the concentration of dNTPs is 100μM to 300μM, preferably 200μM.

[0074] In this invention, the concentration is the final concentration, and more specifically, the usage concentration, which is the concentration when reacting with the target substance.

[0075] The PCR detection procedure was as follows: 55℃ for 5 min; 95℃ for 2 min; 95℃ for 5 s, 60℃ for 30 s, 69℃ for 10 s, 45 cycles; 95℃ for 10 s; 25℃ for 3 min; continuous temperature increase from 25℃ to 68℃ at a rate of 0.03℃ / s.

[0076] In the detection method of the present invention, the "60℃ for 30s" and "25~68℃ continuous temperature increase" stages of PCR detection also include a fluorescence signal detection step; the PCR detection is further followed by a result interpretation step.

[0077] The detection method of the present invention further includes a separate step of detecting positive and negative control samples to achieve quality control. Specifically, the detection of positive and negative control samples includes the following steps: mixing the positive / negative control sample, the reagent (primer-probe mixture) of the present invention, and the PCR reagent (enzyme mixture) in the reagent of the present invention, and then performing PCR detection.

[0078] In specific embodiments of the present invention, the sample, the positive control, and the negative control can be a nucleic acid solution obtained by nucleic acid extraction from a pathogen-containing sample, or it can be a specific pathogen-containing sample that has not undergone nucleic acid extraction; the present invention does not limit this; in the present invention, a nucleic acid solution obtained by nucleic acid extraction from a pathogen-containing sample is preferred;

[0079] In this invention, the detection method includes detection methods for diagnostic purposes and detection methods for non-diagnostic purposes. The detection methods for non-diagnostic purposes include those used in the development of detection products or detection methods to study the impact of changes in specific parameters on the performance of the detection products or to determine the merits of the detection methods.

[0080] In the detection method described in this invention, a single-tube detection method is adopted, and each reaction contains 2 to 6 fluorescence channels, preferably 4; however, after design, this invention can perform 20-fold detection in one tube under the condition that the equipment allows, and detect 18 pathogens.

[0081] The purpose of this invention is to overcome the limitations of existing PCR technology in performing multiplex detection on fluorescent PCR equipment. It provides a primer-probe combination for broad-spectrum screening of respiratory pathogens in common acute and community-acquired pneumonia. This combination offers advantages such as high accuracy, good specificity, strong anti-interference ability, good coverage, high sensitivity, multi-detection capability, and ease of operation. These advantages are the result of the combined effect of various technical parameters. Specifically, it involves screening the gene detection regions of pathogens, designing primer-probe combinations with specific conserved regions as target sequences, and comparing, screening, and evaluating the specificity of conserved sequences and primers. This effectively avoids non-specific amplification and non-specific cross-reactions between multiplex primers, while maintaining high specificity and broad coverage. Furthermore, by optimizing the concentration of the primer-probe combination and screening and optimizing the enzyme mixture components, the sensitivity, anti-interference ability, and specificity of the detection are further improved. Finally, this invention employs a two-probe design, where the interaction between the two probes provides a stable Tm value, thereby further enhancing the specificity and accuracy of multiplex detection results (each Tm value is determined by the corresponding two probes).

[0082] Furthermore, the above optimizations give the present invention the following advantages:

[0083] (1) High specificity: The present invention selects the gene detection region of the pathogen as the specific conserved region, and the gene amplification primers are designed for this conserved sequence. The specificity of the conserved sequence and primers is evaluated by comparison, which effectively avoids non-specific amplification and non-specific cross-reaction between multiple primers. It has high specificity and wide coverage.

[0084] (2) Strong anti-interference ability: The present invention uses clinical samples with added interfering substances (the interfering substances are selected at the potential maximum concentration ("worst condition")) to test the tolerance of interfering substances. The results show that the interfering substances do not interfere with the detection results.

[0085] (3) Good inclusiveness: The present invention can detect the different types of pathogens, indicating that the present invention has good inclusiveness;

[0086] (4) High sensitivity: By optimizing the amount of primers, probes and amplification efficiency of the reaction system, this invention can detect pathogen concentrations of 100 copies / ml;

[0087] (5) Multi-indicator joint detection: This invention can simultaneously detect up to 18 kinds of respiratory pathogens of ARTI and CAP in a single tube, taking into account multiple types such as viruses and atypical pathogens. It has a wide coverage and a wide variety of pathogens, and can provide medical institutions with an accurate, multi-target parallel detection.

[0088] (6) By combining fully automated nucleic acid testing equipment, fully automated testing can be achieved, reducing the possibility of contamination;

[0089] (7) Use of internal standard system: can detect the occurrence of false negatives and avoid false negatives caused by inhibitors in the sample or operational errors;

[0090] (8) Easy to operate and closed-loop testing throughout: No expensive and specialized instruments and equipment are required. The entire process is closed-loop testing without opening the lid, which effectively avoids contamination.

[0091] This invention provides a primer and probe combination and a planned detection method for the broad-spectrum detection of respiratory pathogens in acute and community-acquired pneumonia through optimization of primer and probe sequences, optimization of primer and probe, screening and concentration optimization of components in PCR amplification reagents, and optimization of PCR amplification procedures. This allows for the detection of 20 targets in a single reaction tube and the identification of 18 common respiratory pathogens. It also has advantages such as high accuracy, good specificity, strong anti-interference ability, good inclusiveness, high sensitivity, multi-detection capability, and ease of operation.

[0092] This invention, through screening and optimization, has obtained a primer-probe combination for nucleic acid detection of respiratory pathogens for broad-spectrum screening of common acute respiratory infections and community-acquired pneumonia. The primer-probe combination described in this invention covers 18 respiratory pathogens and has advantages such as high accuracy, good specificity, strong anti-interference ability, good inclusiveness, high sensitivity, and superconducting detection. At the same time, combined with fully enclosed single-tube PCR amplification, it can achieve parallel detection of 20 targets, which greatly improves the early diagnosis of respiratory diseases with complex pathogen structures and is of great significance for the prevention and treatment of infectious diseases. Attached Figure Description

[0093] Figure 1 Showing cross-specific detection results;

[0094] Figure 2 The results of the inclusion assay for adenovirus (HADV) were displayed.

[0095] Figure 3 The results of the inclusive testing for influenza A virus (IFA) are shown.

[0096] Figure 4 This demonstrates the inclusive testing results for the novel coronavirus (SARS-CoV-2);

[0097] Figure 5 The test results showed that all 18 pathogens were positive. Detailed Implementation

[0098] This invention provides primer-probe combinations for broad-spectrum screening of common respiratory pathogens and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0099] Homology comparison analysis was performed on the nucleic acid information of respiratory pathogens to select pathogen-specific conserved sequences and design specific primers and probes, which are shown in Table 1.

[0100] Table 1. Primer sequences for detecting pathogens and internal standards

[0101]

[0102]

[0103] Table 2. Probe sequences for detecting pathogens and internal standards

[0104]

[0105]

[0106] In Table 2, each end (5' end and 3' end) of the fluorescent probe has a fluorescent reporter group (located at the 5' end or 3' end) and a fluorescent quencher group (located at the 5' end or 3' end). In the embodiments, the fluorescent reporter group is one or more of FAM, VIC, ROX and CY5, and the fluorescent quencher group is one or more of BHQ1, BHQ2 and BHQ3. One end (5' segment or 3' end) of the auxiliary probe has a blocking group. In the embodiments, the blocking group is phosphte. Furthermore, in Tables 1 and 2, in addition to the four normal base symbols a, t, g, and c, other letters such as r, y, m, and k also appear in the nucleotide chains. These other letters are degenerate bases, where r represents that the base is a or g, y represents that the base is c or t, m represents that the base is a or c, k represents that the base is g or t, s represents that the base is c or g, w represents that the base is a or t, h represents that the base is a, c, or t, b represents that the base is c, g, or t, v represents that the base is a, c, or g, d represents that the base is a, g, or t, and n represents that the base is a, c, g, or t.

[0107] Table 3. Conserved sequences and internal standard sequences for pathogen detection

[0108]

[0109]

[0110]

[0111] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0112] Example 1: Kit Composition

[0113] This embodiment provides a primer-probe set and its application for broad-spectrum screening of common acute and community-acquired respiratory pathogens, including an enzyme mixture, a primer-probe mixture, DEPC-treated water, a negative control, and a positive control. The enzyme mixture contains reverse transcriptase, an RNase inhibitor, Taq polymerase, PCR buffer, dNTPs, and salt ions. The negative control is a human internal standard gene; the positive control contains amplified sequences for detecting IFB, RSV, CP, and the human internal standard gene.

[0114] The primer-probe mixture includes an amplification primer set and a probe set. The final primer concentration is 100 nM to 300 nM (200 nM is preferred). The final probe concentration is 50 nM to 200 nM (120 nM is preferred).

[0115] Table 4. Final concentrations of major components in the enzyme mixture

[0116] Main ingredients Final concentration reverse transcriptase 1U to 4U (2U is preferred) RNAse inhibitors 5U~20U (10U is better) Taq enzyme 1U to 3U (2U is preferred) dNTPs 100μM~300μM (200μM is better)

[0117] PCR reaction system preparation: (enzyme mixture, primer-probe mixture, DEPC-treated water, nucleic acid template), the formula is shown in Table 5:

[0118] Table 5. Reagent Preparation

[0119] Components Amount added (μL / test) enzyme mixture 4 Primer-probe mixture 3 DEPC treated water 8 Nucleic acid template 5

[0120] PCR amplification program: Stage 1, 55℃, 5min, 1 cycle; Stage 2, 95℃, 2min, 1 cycle; Stage 3, 95℃, 5s, 60℃ (collect fluorescence), 30s, 69℃, 10s, 45 cycles; Stage 4, 95℃, 10s, 25℃, 3min, temperature rise from 25 to 68℃ to collect fluorescence signal, 1 cycle.

[0121] Result Interpretation: The detection channels and melting temperature ranges for each pathogen are shown in Table 6 below. Positive samples should exhibit peak height variations compared to the negative control (NC) melting curve within the corresponding pathogen's detection melting temperature range to determine the specific pathogen type.

[0122] Table 6. Detection channels and melting temperature ranges for various pathogens

[0123]

[0124]

[0125] Example 2: Detection of clinical samples

[0126] Specifically, using the detection method described in Example 1 above, this example presents detection data for clinical samples. The clinical samples are swabs or sputum samples from patients with known infectious agents (ARTI) and community-acquired pneumonia (CAP).

[0127] Nucleic acid was extracted from the samples in Table 7 to obtain nucleic acid templates. The reaction system was prepared according to Table 5 in Example 1, and PCR amplification was performed according to the procedure in Example 1.

[0128] Analysis of the test results revealed 60 positive samples and 20 negative samples. The PCR amplification products from the positive samples were sequenced, and the sequences were input into the NCBI database and compared using BLAST software to confirm whether the pathogen type matched the application's detection results. The results showed that the sequencing results were consistent with the application's detection results, indicating high accuracy of the invention.

[0129] Table 7. Clinical Sample Detection Results

[0130]

[0131]

[0132] Example 3 Sensitivity Test

[0133] The analytical sensitivity test procedure is as follows:

[0134] The clinical samples after the values ​​were determined were diluted to 500, 100, and 50 copies / ml, and nucleic acid was extracted to obtain nucleic acid templates. Specific sample information is shown in Table 8.

[0135] Prepare the reaction system according to Table 5 in Example 1, and perform PCR amplification according to the procedure in Example 1.

[0136] The test was repeated 20 times, and the results are shown in Table 8.

[0137] Table 8. Analytical Sensitivity Sample Information

[0138] Sample Name Concentration (copies / ml) Sample Name Concentration (copies / ml) IFB 500、100、50 OC43 500、100、50 MP 500、100、50 NL63 500、100、50 SARS-CoV-2 500、100、50 HBOV 500、100、50 PIV1 500、100、50 CP 500、100、50 PIV2 500、100、50 HRV 500、100、50 PIV3 500、100、50 HMPV 500、100、50 PIV4 500、100、50 HADV 500、100、50 229E 500、100、50 RSV 500、100、50 HKU1 500、100、50 IFA 500、100、50

[0139] According to the detection results in Table 9, the detection limit of this invention is 100 copies / ml, which indicates high sensitivity.

[0140] Table 9. Statistical Results

[0141]

[0142] Implementation of Case 4: Repeatability Testing

[0143] The procedure for repeatability testing is as follows:

[0144] Each sample in Table 10 is a mixed sample of multiple pathogens, and the types of pathogens contained therein are shown in the "Pathogen Target Combinations"; and all of them have been clinically confirmed.

[0145] Nucleic acid was extracted from the samples in Table 10, the reaction system was prepared according to Table 5 in Example 1, and PCR amplification was performed according to the procedure in Example 1.

[0146] As can be seen from the test results in Table 10, the test method of the present invention has good repeatability.

[0147] Table 10. Test Results

[0148]

[0149]

[0150] Implementation Case 5: Specificity Test

[0151] The specificity testing procedure is as follows:

[0152] Other pathogens besides the 18 pathogens described in this invention were used as templates for detection. The types and concentrations of pathogens in the templates are shown in Table 11.

[0153] Nucleic acid was extracted from the samples in Table 11, the reaction system was prepared according to Table 5 in Example 1, and PCR amplification was performed according to the procedure in Example 1.

[0154] The detection results for pathogens other than the 12 pathogens described in this invention are shown in Table 11. According to the detection results, this invention has high specificity and no cross-reactivity (e.g., ...). Figure 1 (As shown).

[0155] Table 11. Results of Cross-Specificity Detection

[0156]

[0157]

[0158] Implementation Case 6 Anti-interference capability

[0159] The steps for testing anti-interference capability are as follows:

[0160] Nucleic acid extraction was performed after adding interfering substances to samples containing pathogens. The interfering substances were selected based on the potential maximum concentration (“worst condition”). The types of pathogens, interfering substances, and concentrations are detailed in Table 12. The nucleic acid extracted from the pathogen-containing samples with added interfering substances was used as the nucleic acid template.

[0161] Prepare the reaction system according to Table 5 in Example 1, and perform PCR amplification according to the procedure in Example 1.

[0162] The samples with added interfering substances were tested according to the detection method described in the above embodiments. As shown in Table 12, the listed drugs did not interfere with the test results, indicating that the present invention has good anti-interference capability.

[0163] Table 12. Test Results

[0164]

[0165]

[0166]

[0167] Implementation of Case 7: Inclusivity Testing

[0168] The steps for inclusiveness testing are as follows:

[0169] Different pathogen subtypes were used as templates for independent detection. The specific pathogen subtypes are shown in Table 13. All samples were clinically confirmed and used as nucleic acid templates after nucleic acid extraction. Each test detected one specific subtype of pathogen, and the nucleic acid template was obtained by nucleic acid extraction from the pathogen.

[0170] Prepare the reaction system according to Table 5 in Example 1, and perform PCR amplification according to the procedure in Example 1.

[0171] The different types of samples were tested following the steps described above. According to the test results in Table 13, the detection method of this invention can normally detect the listed different types of pathogens, indicating that this invention has good inclusiveness. For example, the inclusiveness detection results for adenovirus (HADV) are shown in the figure below. Figure 2 As shown in the figure, the inclusion test results for influenza A virus (IFA) are as follows. Figure 3 As shown in the figure, the inclusive testing results for the novel coronavirus (SARS-CoV-2) are as follows. Figure 4 As shown.

[0172] Table 13. Test Results

[0173]

[0174]

[0175]

[0176] Example 8 Multiple Tests

[0177] The multi-step testing process is as follows:

[0178] Nucleic acid templates were obtained by extracting nucleic acid from clinically identified mixed pathogens as shown in Table 14 for testing.

[0179] Prepare the reaction system according to Table 5 in Example 1, and perform PCR amplification according to the procedure in Example 1.

[0180] Perform testing on the mixed infection samples following the steps described above.

[0181] According to the test results in Table 13, the detection method of the present invention can normally detect multiple composite infection samples. Figure 5 (Illustrated results of combined detection of 18 pathogens (20 strains)).

[0182] Table 14. Test Results

[0183]

[0184] As demonstrated by the above embodiments, this invention provides a primer and probe set for detecting nucleic acids of common respiratory pathogens in acute and community-acquired pneumonia, and its applications. This set offers advantages such as high specificity, high sensitivity, multiple detection targets, and simple and rapid operation. This invention can simultaneously detect 18 ARTI and CAP respiratory pathogens in a single tube, providing broad coverage and a wide range of pathogens. It can offer CDC centers, hospitals, and other medical institutions an accurate, multi-target parallel detection solution. Combined with fully automated nucleic acid detection equipment, it enables fully automated detection. An internal standard system is included to control false negative results caused by potential PCR inhibitors, instruments, reagents, and operational issues during the detection process. Negative and positive controls are provided to control the quality of the nucleic acid extraction process.

[0185] Example 9 Concentration Optimization

[0186] During the development of this invention, the dosage of each component in the reagent kit was compared and optimized to obtain the optimal set. Table 15 lists some of them exemplarily.

[0187] Table 15. Final concentrations of each component in the comparative test

[0188]

[0189] The testing steps are as follows:

[0190] Clinical samples that tested positive for each pathogen were diluted to two concentration levels (concentration 1: 1.00 × 10⁻⁶).5 copies / mL, concentration 2: 5.00×10 3 Nucleic acid was extracted simultaneously with the negative control (copies / mL); the specific pathogens are shown in Table 15.

[0191] Take the primers and probes from Tables 1 and 2 above and prepare the corresponding three-group primer and probe mixtures 3-5 according to the three groups of final concentration requirements in Table 15; at the same time, prepare the corresponding three-group enzyme mixtures 3-5 according to the final concentration requirements of other components in Table 15.

[0192] Prepare the corresponding PCR reaction systems (3-5 ml of enzyme mixture, 3-5 ml of primer-probe mixture, DEPC-treated water, and nucleic acid template) as shown in Table 5.

[0193] Perform PCR amplification according to the procedure in Example 1.

[0194] The three reaction systems were tested twice for each concentration level of the sample, and the Ct values ​​are shown in Table 16.

[0195] Table 16. Summary of Ct values ​​for comparative detection

[0196]

[0197]

[0198] Comparison Case 1

[0199] During the development of this invention, multiple primer and probe sets were designed for 18 pathogens, and the optimal set was obtained through comparative experiments. For brevity, Table 17 lists a portion of these sets.

[0200] Table 17. Primer sequences for detecting pathogens and internal standards

[0201]

[0202]

[0203]

[0204] The screening test steps are as follows:

[0205] Clinical samples positive for each pathogen were diluted to three concentration levels (high, medium, and low), and nucleic acid was extracted simultaneously with negative controls; the high, medium, and low concentration levels each represented a concentration of 1.00 × 10⁻⁶. 6 copies / mL, 1.00×10 5 copies / mL, 1.00×10 4copies / mL; specific pathogen types are shown in Table 18, and nucleic acid templates were obtained through nucleic acid extraction;

[0206] The primers and probes in Tables 1 and 2 above were used to prepare primer and probe mixture 1, and the primers and probes in Table 17 were used to prepare primer and probe mixture 2.

[0207] Prepare the corresponding PCR reaction systems (enzyme mixture, primer-probe mixture 1 or primer-probe mixture 2, DEPC-treated water, and nucleic acid template) as shown in Table 5.

[0208] Perform PCR amplification according to the procedure in Example 1.

[0209] The two reaction systems were tested three times for each concentration level, and the Ct values ​​are shown in Table 18. The corresponding Tm values ​​for each target are shown in Table 19.

[0210] Table 18. Summary of Ct values ​​for comparative detection

[0211]

[0212]

[0213]

[0214] Table 19. Summary of Tm values ​​for comparative testing

[0215]

[0216] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications 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.

Claims

1. A primer-probe combination for detecting respiratory pathogens, characterized in that, The primer-probe combination includes: A primer set and probe for detecting influenza B virus, comprising: an upstream primer with the nucleotide sequence shown in SEQ ID NO. 1, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 2, and probes with the nucleotide sequences shown in SEQ ID NO. 41 and SEQ ID NO. 42; and A primer set and probe for the detection of Mycoplasma pneumoniae, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 3, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 4, and probes with nucleotide sequences as shown in SEQ ID NO. 43 and SEQ ID NO. 44; and A primer set and probe for detecting the novel coronavirus, comprising: primer set and probe combination 1 and primer set and probe combination 2; primer set and probe combination 1 comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 5, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 6, and probes with nucleotide sequences as shown in SEQ ID NO. 45 and SEQ ID NO. 46; primer set and probe combination 2 comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 35, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 36, and probes with nucleotide sequences as shown in SEQ ID NO. 75 and SEQ ID NO. 76; and A primer set and probe for detecting parainfluenza virus type 1, comprising: an upstream primer with the nucleotide sequence shown in SEQ ID NO. 7, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 8, and probes with the nucleotide sequences shown in SEQ ID NO. 47 and SEQ ID NO. 48; and A primer set and probe for detecting parainfluenza virus type 2, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 9, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 10, and probes with nucleotide sequences as shown in SEQ ID NO. 49 and SEQ ID NO. 50; and A primer set and probe for detecting parainfluenza virus type 3, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 11, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 12, and probes with nucleotide sequences as shown in SEQ ID NO. 51 and SEQ ID NO. 52; and A primer set and probe for detecting parainfluenza virus type 4, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 13, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 14, and probes with nucleotide sequences as shown in SEQ ID NO. 53 and SEQ ID NO. 54; and A primer set and probe for the detection of coronavirus 229E, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 15, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 16, and probes with nucleotide sequences as shown in SEQ ID NO. 55 and SEQ ID NO. 56; and A primer set and probe for the detection of coronavirus HKU, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 17, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 18, and probes with nucleotide sequences as shown in SEQ ID NO. 57 and SEQ ID NO. 58; and A primer set and probe for the detection of coronavirus OC43, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 19, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 20, and probes with nucleotide sequences as shown in SEQ ID NO. 59 and SEQ ID NO. 60; and A primer set and probe for the detection of coronavirus NL63, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 21, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 22, and probes with nucleotide sequences as shown in SEQ ID NO. 61 and SEQ ID NO. 62; and A primer set and probe for bocavirus detection, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 23, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 24, and probes with nucleotide sequences as shown in SEQ ID NO. 63 and SEQ ID NO. 64; and A primer set and probe for the detection of Chlamydia pneumoniae, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 25, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 26, and probes with nucleotide sequences as shown in SEQ ID NO. 65 and SEQ ID NO. 66; and A primer set and probe for rhinovirus detection, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 27, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 28, and probes with nucleotide sequences as shown in SEQ ID NO. 67 and SEQ ID NO. 68; and A primer set and probe for the detection of metapneumovirus, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 29, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 30, and probes with nucleotide sequences as shown in SEQ ID NO. 69 and SEQ ID NO. 70; and A primer set and probe for adenovirus detection, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 31, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 32, and a probe with nucleotide sequences as shown in SEQ ID NO. 71 and SEQ ID NO. 72; and A primer set and probe for respiratory syncytial virus (RSV) detection, comprising: an upstream primer with the nucleotide sequence shown in SEQ ID NO. 33, a downstream primer with the nucleotide sequence shown in SEQ ID NO. 34, and a probe with the nucleotide sequences shown in SEQ ID NO. 73 and SEQ ID NO. 74; and A primer set and probe for detecting influenza A virus, the primer set and probe comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 37, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 38, and a probe with nucleotide sequences as shown in SEQ ID NO. 77 and SEQ ID NO.

78.

2. The primer-probe combination according to claim 1, characterized in that, The primer-probe combination also includes primer sets and probes for detecting internal controls; The primer set and probes used for detecting the internal reference include: an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 39, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 40, and probes with nucleotide sequences as shown in SEQ ID NO. 79, SEQ ID NO. 80, and SEQ ID NO.

81.

3. A reagent, characterized in that, Includes the primer-probe combination as described in claim 1 or 2.

4. A reagent kit, characterized in that, Includes the primer-probe combination as described in claim 1 or 2, or the reagent as described in claim 3, and PCR detection reagents.

5. The reagent kit according to claim 4, characterized in that, The PCR detection reagents include DNA polymerase, reverse transcriptase, RNase inhibitors, and / or dNTPs.

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