Composition for detecting respiratory tract infection related pathogens, kit and application

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of respiratory tract infection related pathogens, and more particularly relates to detection of Huofen dystrophy, Nocardia, Moraxella catarrhalis and Chlamydia psittaci. According to the combined detection composition provided by the invention, different pathogens are detected by mainly utilizing a multiple fluorescent PCR analysis method and detecting target spots on the different pathogens, so that detection and distinguishing of Huofencur dystrophe, Nocardia, Moraxella catarrhalis and Chlamydia psittaci are simultaneously realized in a single-tube reaction system; therefore, a targeted strategy is provided for subsequent treatment. The composition disclosed by the invention is higher in detection sensitivity (up to 200 copies / mL), good in specificity and more accurate in detection, provides a more sufficient basis for rapid diagnosis and exclusion of infection of different pathogens for clinicians, shortens the time for the clinicians to diagnose the condition of a patient, and accelerates the implementation of treatment measures on the patient.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of pathogens related to respiratory tract infection, and more specifically, to the detection of Trophobia whipplei, Nocardia, Moraxella catarrhalis, and Chlamydia psittaci. Background Art

[0002] Tropheryma Whipplei (TW) is a conditionally pathogenic bacterium that can cause a rare disease, Whipple's disease (WD). TW is a relatively rare pathogen that can cause infections that affect multiple organs, with diverse and nonspecific symptoms that are easily ignored by clinicians. In addition, people have a lack of understanding of WD and limited diagnostic methods, so the disease is often misdiagnosed.

[0003] Nocardia is a Gram-positive, aerobic filamentous bacterium with weak acid resistance. It is widely distributed in soil, saprophytes and water. It can enter the body through the respiratory tract, broken skin, wounds, and digestive tract. When the body's resistance is reduced for various reasons, it can cause disease. After a person is infected with Nocardia, the clinical manifestations lack characteristic features and often manifest as acute, subacute, chronic localized or disseminated suppurative diseases, with symptoms such as fever, cough, sputum, and chest tightness. Involvement of the pleura may cause chest pain, and some may develop pleural effusions, most of which are purulent. A very small number of cases will form fistulas connected to the bronchus or chest wall, thereby forming purulent pneumothorax. In recent years, due to the widespread use of hormones and immunosuppressants, the incidence of Nocardia has been on the rise.

[0004] Moraxella catarrhalis is a gram-negative diplococcus that lives in the human respiratory tract. It is often in the form of kidney-shaped diplococci in sputum and exists inside or outside phagocytes. It is generally non-pathogenic. When the body's immunity is low, it can cause mucosal catarrhal inflammation, acute pharyngitis, bronchitis, pneumonia, acute otitis media or meningitis alone or with other bacteria. It is a common pathogen that causes respiratory tract infections in patients in hospitals.

[0005] Chlamydia psittaci is a Gram-negative zoonosis pathogen that is an obligate eukaryotic intracellular parasite with a unique two-phase development cycle. Chlamydia psittaci can infect humans through the respiratory tract in the form of air or aerosols, and can also infect human skin, mucous membranes and digestive tract through excrement, causing mild flu-like symptoms to life-threatening atypical community-acquired pneumonia. The onset of psittacosis is often insidious, and severe patients may develop acutely, with an incubation period of 5 to 14 days, or as long as 21 days. If it is not diagnosed in time, there will be a greater risk.

[0006] Trophophaga whipplei, Nocardia, Moraxella catarrhalis, and Chlamydia psittaci can all cause human respiratory tract infections, mainly causing symptoms such as fever, cough, pharyngitis, and headache, and the clinical symptoms are atypical. Severe cases can lead to the occurrence of atypical pneumonia, and then to a variety of complications. Therefore, there is a need in the art for a product that can simply and quickly detect the above pathogens, and has good sensitivity and specificity, and can detect and distinguish pathogenic microorganisms that cause respiratory tract infections, so as to provide targeted strategies for the prevention and treatment of the above infections. Summary of the invention

[0007] In view of this, in a first aspect, the present invention provides a composition for detecting pathogens related to respiratory tract infection, comprising:

[0008] Upstream primers, downstream primers and probes for detecting Trochophora whipplei as shown in SEQ ID NOs: 1 to 3;

[0009] Upstream primers, downstream primers and probes for detecting Nocardia as shown in SEQ ID NOs: 4 to 6;

[0010] Upstream primers, downstream primers and probes for detecting Moraxella catarrhalis as shown in SEQ ID NOs: 7 to 9; and

[0011] The upstream primers, downstream primers and probe for detecting Chlamydia psittaci are shown in SEQ ID NOs: 10-12.

[0012] The combined detection composition provided by the present invention mainly utilizes a multiplex fluorescent PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and differentiation of Whipple's Trophobia, Nocardia, Moraxella catarrhalis, and Chlamydia psittaci in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. The composition of the present invention has a higher detection sensitivity of 200 copies / mL, good specificity, and more accurate detection, providing clinicians with a relatively sufficient basis for rapid diagnosis and exclusion of different pathogen infections, shortening the time for clinicians to diagnose the patient's condition, and accelerating the implementation of treatment measures for the patient.

[0013] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0014] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0015] In a specific embodiment, the internal standard is RNase P or NADPH.

[0016] Furthermore, the fluorescent groups of the probes used in the composition of the present invention to detect different pathogens are different from each other and do not interfere with each other.

[0017] 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 and CY5 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.

[0018] In some specific embodiments, the fluorescent reporter group of the probe for detecting Tropical Cyst whipplei is FAM; the fluorescent reporter group of the probe for detecting Nocardia is HEX (or VIC); the fluorescent reporter group of the probe for detecting Moraxella catarrhalis is ROX; and the fluorescent reporter group of the probe for detecting Chlamydia psittaci is CY5.

[0019] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe pairs. In the present invention, "set" refers to upstream and downstream primers and probes that match each other for detecting a target.

[0020] The composition of the present invention can be combined into any combination of the corresponding four targets. Those skilled in the art can combine as needed to detect which targets to detect, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0021] For example, any three of the four sets of primers and probes may be included, any two of the four sets of primers and probes may be included, or any one of the four sets of primers and probes may be included.

[0022] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0023] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0024] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0025] In a specific embodiment, the 3' end of the probe is BHQ1.

[0026] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0027] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0028] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0029] 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 pathogen is one or more of Chlamydia psittaci, Tropical Whipple, Nocardia, or Moraxella catarrhalis.

[0030] 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.

[0031] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0032] In a specific embodiment, the negative control is at least one of DEPC H2O and normal saline; the positive control is at least one of a fragment plasmid or a positive strain of Chlamydia psittaci, Tropical Whipple, Nocardia, and Moraxella catarrhalis.

[0033] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0034] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG).

[0035] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0036] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UNG), DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0037] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0038] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UDG enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.

[0039] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0040] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0041] In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.

[0042] In a fourth aspect, a method for detecting pathogens associated with respiratory tract infection is provided, the method comprising the following steps:

[0043] 1) Extracting nucleic acid from the sample to be tested;

[0044] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0045] 3) Obtain and analyze the results.

[0046] In the present invention, the sample used for detection may be sputum, respiratory secretions, etc., but is not limited thereto.

[0047] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0048] UNG enzyme reaction, temperature is 50-60℃, time is 0.5-6min, 1 cycle; Taq enzyme activation, temperature is 90-95℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-95℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0049] Furthermore, the method for detecting pathogens associated with respiratory tract infection is for non-diagnostic purposes.

[0050] In a specific embodiment, a composition is provided for preparing a reagent for detecting pathogens associated with respiratory tract infection, wherein the detection comprises the following steps:

[0051] 1) Extracting nucleic acid from the sample to be tested;

[0052] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0053] 3) Obtain and analyze the results.

[0054] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0055] UNG enzyme reaction, temperature is 50-60℃, time is 0.5-6min, 1 cycle; Taq enzyme activation, temperature is 90-95℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-95℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0056] 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 pathogens and suffers from the infection. For example, the method can detect whether the environment, culture, and drinking water sources have the above pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a graph showing the test results of the composition of the present invention (Trophycopsis Whipple, Nocardia, Moraxella catarrhalis, Chlamydia psittaci);

[0058] Figures 2 to 5 Graph showing the sensitivity results of the composition of the present invention (respectively Trophicola Whipple, Nocardia, Moraxella catarrhalis, and Chlamydia psittaci);

[0059] Figure 6 It is a specific result diagram of the composition of the present invention;

[0060] Figures 7-10 The anti-interference result diagram of the composition of the present invention (respectively Trophicola Whipple, Nocardia, Moraxella catarrhalis, Chlamydia psittaci);

[0061] Fig.11 This is a graph showing the test results of the comparative example composition of the present invention. DETAILED DESCRIPTION

[0062] 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.

[0063] Example 1: Primers and probes used in the present invention

[0064] The primers and probes used in the present invention are shown in Table 1 below:

[0065] Table 1

[0066]

[0067]

[0068] Among them, the fluorescent reporter group of the Whipple's Trophobia probe is FAM; the fluorescent reporter group of the Nocardia probe is HEX; the fluorescent reporter group of Moraxella catarrhalis is ROX; and the fluorescent reporter group of the Chlamydia psittaci probe is CY5.

[0069] Example 2: Method for detecting pathogens

[0070] Reagent preparation:

[0071] Preparation of enzyme mixture:

[0072] The DNA polymerase mixture is composed of UNG and H-Taq enzymes, which are prepared by mixing H-Taq enzyme (5U / μL) and UNG enzyme (2U / μL) in a certain ratio.

[0073] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution and enzyme mixture, mix them thoroughly to form a PCR mixture, centrifuge at 2000 rpm for 10 seconds and set aside. The real-time fluorescence PCR reaction system is configured according to the following Table 2.

[0074] Table 2

[0075] Components Volume / concentration in each reaction PCR Buffer 22.95μL UNG enzyme 0.5μL (2U / μL) Taq enzyme 3.5μL (5U / μL) Mg2+ 0.15μL(1M) dNTP 0.5μL(25mM) Primers 1.6μL (25pmol / uL) Probe 0.8μL (25pmol / uL) Sample DNA 20μL

[0076] Sample handling and loading:

[0077] Take 200 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and use the nucleic acid extraction or purification reagent of Shengxiang Biotechnology Co., Ltd. to extract nucleic acid according to its instructions; take 5 μL of the above-treated samples, negative controls, and positive controls and add them to the corresponding 0.2 mL PCR reaction tubes, add 20 μL of PCR mixture to each tube, and cover the tube.

[0078] PCR amplification:

[0079] PCR amplification was performed on a SLAN-96P real-time fluorescence quantitative PCR instrument according to the temperature and time settings shown in Table 3.

[0080] Table 3

[0081]

[0082] Result analysis and judgment

[0083] After the reaction is completed, the results are automatically saved, and the amplification curves of the detection targets are analyzed separately. According to the analyzed image, adjust the Start value, End value, and Threshold value of the Baseline (users can adjust according to actual conditions, the Start value can be set between 3 and 15, and the End value can be set between 5 and 20, and adjust the amplification curve of the negative control to make it flat or below the threshold line), click Analyze to analyze, so that each parameter meets the requirements of the following "Quality Control", and then record the qualitative results in the Plate window.

[0084] Quality Control:

[0085] If the sample has an obvious S-shaped amplification curve in the FAM, HEX (VIC), ROX, CY5 channels and the Ct value is ≤40, it is judged as positive; if the sample has no amplification curve (No Ct) or the Ct value is greater than 40 in the FAM, HEX (VIC), ROX, CY5 channels, it is judged as negative. The details are shown in Table 4 below.

[0086] Table 4

[0087]

[0088]

[0089] Example 3: Test results of the test samples of the composition of the present invention

[0090] The primers and probes shown in Example 1 were used to perform PCR detection on a mixed sample of T. whipplei, Nocardia, Moraxella catarrhalis, and Chlamydia psittaci on a SLAN-96P real-time fluorescence quantitative PCR instrument according to the method of Example 2. The detection results are shown in FIG. Figure 1 As shown in Table 5, it can be seen from the figure that the composition of the present invention can perform good detection on various pathogen combinations.

[0091] Table 5

[0092] Whipple's Trophic Body Nocardia Moraxella catarrhalis Chlamydia psittaci Sample 1 33.93 31.27 27.82 30.8 Sample 2 29.77 28.48 25.21 28.35 Sample 3 33.84 35.65 28.46 31.66 Sample 4 30.57 28.29 24.68 28.15 Sample 5 32.36 31.61 28.18 31.87

[0093] Example 4: Sensitivity of the composition of the present invention

[0094] Using the composition in Example 1 of the present invention, a gradient dilution of the plasmid was taken, and the concentrations were 500000, 50000, 5000, 500, and 200 copies / mL, respectively, and the five concentrations were tested. The results are as follows Figures 2 to 5 As shown, it is shown that each channel can still accurately detect samples as low as 200 copies / mL, with a detection rate of 100%, indicating that the sensitivity of the composition of the present invention is 200 copies / mL.

[0095] Example 5. Specificity of the composition of the present invention

[0096] The composition of the present invention has no cross-reaction with other pathogens similar to pathogens related to respiratory tract infections (new coronavirus, influenza A virus, influenza B virus, adenovirus, human immunodeficiency virus type 1, Escherichia coli, Pseudomonas aeruginosa, human parainfluenza virus type 3, cytomegalovirus, coxsackie virus type A, Bordetella pertussis, Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis). The results are as follows Figure 6 As shown, it is shown that the composition of the present invention has good specificity.

[0097] Example 6: Anti-interference and stability of the composition of the present invention

[0098] The composition of Example 1 was analyzed by adding interfering substances. The experimental results showed that potential PCR inhibitors / interfering substances such as 50 μg / mL dexamethasone, 100 μg / mL ribavirin, 100 μg / mL azithromycin, 320 μg / mL budesonide, 125 μg / mL phenylephrine, and 100 μg / mL lopinavir had no obvious effect on the kit. The results are shown in Figures 7 to 10.

[0099] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0100] 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.

[0101] Therefore, the inventors also designed other primers and probes to form different detection systems (sequences are shown in Table 6), which are also used to detect the above pathogens. Fig.11 As shown in the figure, it can be seen that the results show that only part of the amplification curve appears in the detection, and the amplification curve has a low increase and poor repeatability, while other targets do not even have amplification curves. Therefore, the overall detection effect is poor.

[0102] Table 6

[0103]

[0104]

Claims

1. A composition for detecting pathogens associated with respiratory tract infection, comprising: Upstream primers, downstream primers and probes for detecting Trochophora whipplei as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Nocardia as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Moraxella catarrhalis as shown in SEQ ID NOs: 7 to 9; and, The upstream primers, downstream primers and probe for detecting Chlamydia psittaci are shown in SEQ ID NOs: 10-12.

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

3. The composition according to claim 2, characterized in that The fluorescent reporter group of the probe for detecting Trochophora whipplei is FAM; the fluorescent reporter group of the probe for detecting Nocardia is HEX or VIC; the fluorescent reporter group of the probe for detecting Moraxella catarrhalis is ROX; and the fluorescent reporter group of the probe for detecting Chlamydia psittaci is CY5.

4. The composition according to any one of claims 1 to 3, characterized in that The composition also includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

5. The composition according to claim 4, characterized in that The components of the composition are present in admixture.

6. Use of the composition according to any one of claims 1 to 5 in preparing a kit for detecting pathogens related to respiratory tract infection, wherein: The pathogen is one or more of Chlamydia psittaci, Trophicola whipplei, Nocardia, or Moraxella catarrhalis.

7. A kit for detecting pathogens associated with respiratory tract infection, the kit comprising the composition according to any one of claims 1 to 5.

8. The kit according to claim 7, characterized in that The kit also includes a negative quality control product and a positive quality control product.

9. The kit according to claim 7 or 8, characterized in that The kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a DNA polymerase, dNTP, dUTP, UNG enzyme, a PCR buffer, or Mg 2+ At least one of .

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 PCR detection on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 5 or the kit according to any one of claims 7 to 9; 3) Obtain and analyze the results.

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