Composition for detecting opportunity infection related pathogens, kit and application

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of opportunity infection related pathogens, and more particularly relates to detection of Rosella airborne, Rosella mucilaginosa, Rosella saprocaries and Nocardia. 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 Rosella aerogenes, Rosella mucilaginosa, Rosella caries and Nocardia are realized in a single-tube reaction system at the same time, and a targeted strategy is provided for subsequent treatment. The composition provided by the invention has the advantages of higher detection sensitivity (up to 500 copies / mL), good specificity and more accurate 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 opportunistic infections, and more specifically, to the detection of Roseburia aeruginosa, Roseburia mucosa, Roseburia dentalis, and Nocardia. Background Art

[0002] Roseburia belongs to the Micrococcaceae family and is an irregular rod with an optimal growth temperature of 35-37°C. It usually parasitizes in the human mouth and throat and is a conditional pathogen. There are currently 14 species under it, and Rothia dentocariosa, Rothia mucilaginosa and Rothia aeria are Roseburia associated with clinical infections.

[0003] Rothia aeria is a rare human pathogen. The related infections caused are commonly bacteremia, neck abscess, respiratory tract infection, infectious arthritis, infectious endocarditis, etc. The bacteria are Gram-positive, branched, filamentous or radial, without spores or capsules, and negative for acid-fast staining. The colonies grow irregularly, with wrinkles on the surface, opaque, raised, and shiny around. Rothia aeria is now clearly defined as a fixed bacterium in the upper gastrointestinal tract. In addition to the common oral cavity, it is also possible to have intestinal translocation. Due to the rarity of Rothia aeria and the incomplete identification scheme, its risk factors have not been well confirmed.

[0004] Rothia dentocariosa is a Gram-positive bacterium with no fixed morphology. The colonies are raised, highly curled, and present as filamentous morphology, or round, raised, smooth-surfaced, and neatly-margined colonies, presenting the morphology of bacilli or cocci, milky white or occasionally charcoal black, neat, and soft. Rothia dentocariosa is an opportunistic pathogen that is commonly seen in infective endocarditis and can also cause intra-abdominal infections, pneumonia, perivalvular abscesses, endophthalmitis, and bacterial aneurysms.

[0005] Rothia mucilaginosa is a Gram-positive bacterium with spherical bodies, mostly arranged in pairs or quadruplets, and also arranged in grape-like forms; it is a grayish white, gum-like colony with round protrusions, neat and smooth edges, and non-hemolytic; Rothia mucilaginosa is a normal flora of the human nasopharynx, oropharynx, and upper respiratory tract, and can be isolated from nasopharyngeal and bronchial secretions. However, this bacterium can cause various infections in humans, such as endocarditis, bacteremia, peritonitis, sepsis, etc., and is an important opportunistic pathogen.

[0006] Nocardia (Nocardia.N) is a Gram-positive bacterium belonging to the Nocardaceae family and the Nocardia genus. The bacteria have well-developed branched hyphae, similar to actinomycetes, but the ends of the hyphae are not swollen. Nocardia is widely present in soil, seawater, fresh water, and dust. Nocardia is an opportunistic pathogen that mainly infects patients with weakened immune function or complications, infects the human body through the respiratory tract or wounds, and causes pulmonary nocardiosis, cutaneous nocardiosis, endocarditis, intestinal nocardiosis, etc. In severe cases, it can cause nervous system infection and endanger the lives of humans and animals.

[0007] People with low immunity who are not related to diseases caused by airborne Roseburia, dental caries Roseburia, mucus Roseburia, and Nocardia may develop corresponding symptoms, and the clinical symptoms are complex and diverse, and may lead to death of patients in severe cases. 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 opportunistic infections, so as to provide targeted strategies for the prevention and treatment of the above infections. Summary of the invention

[0008] In view of this, in a first aspect, the present invention provides a composition for detecting pathogens related to opportunistic infections, comprising:

[0009] Upstream primers, downstream primers and probes for detecting Roseburia aeruginosa as shown in SEQ ID NOs: 1 to 3;

[0010] Upstream primers, downstream primers and probes for detecting Roseburia mucilaginosa as shown in SEQ ID NOs: 4 to 6;

[0011] Upstream primers, downstream primers and probes for detecting Roseburia dentata as shown in SEQ ID NOs: 7 to 9; and

[0012] The upstream primers, downstream primers and probe for detecting Nocardia are shown in SEQ ID NOs: 10-12.

[0013] 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 air Roseburia, mucus Roseburia, caries Roseburia, and Nocardia 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 500 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.

[0014] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0015] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0016] In a specific embodiment, the internal standard is RNase P or NADPH.

[0017] 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.

[0018] 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.

[0019] In some specific embodiments, the fluorescent reporter group of the probe for detecting air Roseburia is FAM; the fluorescent reporter group of the probe for detecting mucus Roseburia is HEX (or VIC); the fluorescent reporter group of the probe for detecting caries Roseburia is ROX; and the fluorescent reporter group of the probe for detecting Nocardia is CY5.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0024] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0025] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0026] In a specific embodiment, the 3' end of the probe is BHQ1.

[0027] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0028] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0029] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0030] In a second aspect, the present invention provides a use of the above-mentioned composition of the present invention in preparing a kit for detecting pathogens related to opportunistic infections, wherein the pathogen is one or more of Nocardia, Roseburia aeruginosa, Roseburia mucis, or Roseburia dentata.

[0031] In a third aspect, the present invention provides a kit for detecting pathogens associated with opportunistic infections, the kit comprising the composition of the present invention as described above.

[0032] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0033] 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 Nocardia, Roseburia aerogenes, Roseburia mucosa, and Roseburia dentata.

[0034] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0035] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG).

[0036] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0037] 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 .

[0038] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0039] 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.

[0040] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0041] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0042] 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.

[0043] In a fourth aspect, a method for detecting pathogens associated with opportunistic infections is provided, the method comprising the following steps:

[0044] 1) Extracting nucleic acid from the sample to be tested;

[0045] 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);

[0046] 3) Obtain and analyze the results.

[0047] In the present invention, the sample used for detection may be blood, etc., but is not limited thereto.

[0048] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0049] UNG enzyme reaction, temperature is 50-60℃, time is 1-6min, 1 cycle; Taq enzyme activation, temperature is 90-95℃, time is 1-6min, 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.

[0050] Furthermore, the method for detecting pathogens associated with opportunistic infections is for non-diagnostic purposes.

[0051] In a specific embodiment, a composition is provided for use in preparing a reagent for detecting pathogens associated with opportunistic infections, wherein the detection comprises the following steps:

[0052] 1) Extracting nucleic acid from the sample to be tested;

[0053] 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);

[0054] 3) Obtain and analyze the results.

[0055] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0056] UNG enzyme reaction, temperature is 50-60℃, time is 1-6min, 1 cycle; Taq enzyme activation, temperature is 90-95℃, time is 1-6min, 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.

[0057] 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

[0058] Figure 1 This is a graph showing the test results of the composition of the present invention (airy Roseburia, mucus Roseburia, caries Roseburia, and Nocardia);

[0059] Figures 2 to 5 is a sensitivity result diagram of the composition of the present invention (Rocheariae aeruginosa, Rocheariae mucus, Rocheariae dentalis, and Nocardia spp.);

[0060] Figure 6 It is a specific result diagram of the composition of the present invention;

[0061] Figures 7-10 is a graph of the precision results of the composition of the present invention (Rocheariae aeruginosa, Rocheariae mucus, Rocheariae dentalis, and Nocardia spp.);

[0062] Figures 11 to 14 The anti-interference results of the composition of the present invention are shown in Figure 1 (Rocheariae aeruginosa, Rocheariae mucus, Rocheariae dentalis, and Nocardia);

[0063] Fig.15 This is a graph showing the test results of the comparative example composition of the present invention. DETAILED DESCRIPTION

[0064] 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.

[0065] Example 1: Primers and probes used in the present invention

[0066] The primers and probes used in the present invention are shown in Table 1 below:

[0067] Table 1

[0068]

[0069]

[0070] Among them, the fluorescent reporter group of the air Roseburia probe is FAM; the fluorescent reporter group of the mucus Roseburia probe is HEX; the fluorescent reporter group of the caries Roseburia is ROX; and the fluorescent reporter group of the Nocardia probe is CY5.

[0071] Example 2: Method for detecting pathogens

[0072] Reagent preparation:

[0073] Preparation of enzyme mixture:

[0074] 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.

[0075] 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.

[0076] Table 2

[0077] Components Volume / concentration in each reaction dNTPs(100mM) 0.2mM <![CDATA[1mol / L MgCl2]]> 4mM UNG enzyme (2U / μl) 0.1U Taq enzyme (5U / μl) 5U Primers 200nM Probe 100nM PCR buffer (1x) Make up to 45 μL

[0078] Sample handling and loading:

[0079] 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.

[0080] PCR amplification:

[0081] 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.

[0082] Table 3

[0083]

[0084] Result analysis and judgment

[0085] 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.

[0086] Quality Control:

[0087] If the sample has an obvious S-shaped amplification curve in the FAM, HEX (VIC), ROX, and 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, ROX, and CY5 channels, and the HEX (VIC) internal standard channel is positive (Ct value ≤40), it is judged as negative. The details are shown in Table 4 below.

[0088] Table 4

[0089]

[0090]

[0091] Example 3: Test results of the test samples of the composition of the present invention

[0092] The primers and probes shown in Example 1 were used to perform PCR detection on a mixed sample of air Roseburia, mucus Roseburia, caries Roseburia, and Nocardia in accordance with the method of Example 2 on a SLAN-96P real-time fluorescence quantitative PCR instrument. The detection results are shown in FIG. Figure 1 As shown in the figure, it can be seen that the composition of the present invention can perform good detection on various pathogen combinations.

[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 500 copies / mL, with a detection rate of 100%, indicating that the sensitivity of the composition of the present invention is 500 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 opportunistic infection-related pathogens, such as hemolytic Staphylococcus, epidermidis Staphylococcus, baumannii, Escherichia coli, Serratia marcescens, Stenotrophomonas maltophilia, Enterococcus faecalis, Candida albicans, Klebsiella oxytoca, Streptococcus pyogenes, Micrococcus luteus, Rhodococcus equi, Listeria monocytogenes, Acinetobacter junii, Haemophilus parainfluenzae, Legionella dumov, Enterobacter aerogenes, Haemophilus hemolyticus, Oligosaccharomyces maltophilia, Mycoplasma pneumoniae, Chlamydia pneumoniae, Streptococcus salivarius, Neisseria meningitidis, Mycobacterium tuberculosis, influenza A virus, influenza B virus, Aspergillus fumigatus, Aspergillus terreus, Aspergillus fumigatus, Candida glabrata, and Candida tropicalis. The results are as follows Figure 6 As shown (the starting peak is the positive control), it shows that the composition of the present invention has good specificity.

[0097] Example 6: Precision of the composition of the present invention

[0098] Two concentration levels (500,000 copies / ml and 5,000 copies / ul) of quality control products were selected for precision determination, and each sample was repeated 10 times. The results showed that the detection rate of the two reference products was 100%, and the coefficient of variation (CV) of the detection Ct value was less than 5%. Figures 7-10 As shown in the result chart, it can be seen that this kit has good detection precision.

[0099] Example 7. Anti-interference and stability of the composition of the present invention

[0100] The composition of Example 1 was analyzed for added interferences. The experimental results showed that potential PCR inhibitors / interfering substances such as 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, 10 μg / mL heme, and 20 μg / mL purified mucin had no significant effect on the kit. The results are as follows: Figures 11 to 14 shown.

[0101] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0102] 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.

[0103] Therefore, the inventors also designed some other primers and probes to form different detection systems (sequences not shown), which are also used to detect the above pathogens. Fig.15 As shown in the figure, it can be seen that after the joint detection reagent is formed, the fluorescence increment of each target is very different, and the curve shape is worse than that of the composition of the present invention, and does not reach the expected S-type. At the same time, its Ct value is also delayed compared with the composition of the present invention, which shows that the amplification efficiency is not good, far inferior to the composition of the present invention, and even some channels do not produce lines, indicating that the amplification of this channel is seriously affected.

Claims

1. A composition for detecting pathogens related to opportunistic infections, comprising: Upstream primers, downstream primers and probes for detecting Roseburia aeruginosa as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Roseburia mucilaginosa as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Roseburia dentata as shown in SEQ ID NOs: 7 to 9; and, The upstream primers, downstream primers and probe for detecting Nocardia 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 air Roseburia is FAM; the fluorescent reporter group of the probe for detecting mucus Roseburia is HEX or VIC; the fluorescent reporter group of the probe for detecting caries Roseburia is ROX; and the fluorescent reporter group of the probe for detecting Nocardia 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 opportunistic infections, wherein: The pathogen is one or more of Nocardia, Roseburia aeruginosa, Roseburia mucosa, or Roseburia dentata.

7. A kit for detecting pathogens related to opportunistic infections, 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 related to opportunistic infections, 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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