Composition for detecting intestinal fungal infection and monitoring flora repair

Abstract

The invention belongs to the field of molecular biological detection, and provides a joint detection composition which mainly utilizes a multiple fluorescent PCR analysis method to detect related pathogens, so that at least four of bifidobacterium, lactobacillus, candida tropicalis, fusarium, tritirachium album and mucor are detected and distinguished in a single-tube reaction system at the same time. Meanwhile, the detection sensitivity of the composition is higher and reaches 200 copies / mL, and the detection is more accurate.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically relates to a composition for detecting intestinal fungal infection and flora repair monitoring, such as detection and differentiation of bifidobacteria, lactobacilli, tropical Candida, Fusarium, Candida albicans, and Mucor. Background Art

[0002] Chronic diarrhea is one of the common complications of AIDS. Studies have shown that in addition to protozoan infection and viral infection, bacterial and fungal infections are also very important causes of diarrhea in AIDS patients. Among them, tropical Candida, Fusarium, Candida albicans, and Mucor are common causes of diarrhea caused by fungal infection in AIDS patients.

[0003] AIDS patients have disordered immune function, which will be further disturbed after using antibiotics and antibacterial drugs. Therefore, caution should be exercised when using antibacterial drugs on such patients in clinical practice. Bifidobacteria in the intestines are beneficial bacteria that provide nutrition and immune regulation; Lactobacillus is a beneficial bacteria that improves the body's immune ability; the use of antibacterial drugs in my country is relatively rampant, and some people often continue to use antibacterial drugs when they have persistent diarrhea, which reduces their own immune function and causes an imbalance in the ratio of beneficial and harmful bacteria in their bodies. At this time, patients are more susceptible to fungal infection. Therefore, patients with AIDS-related chronic diarrhea should not blindly use antifungal drugs for treatment. If a large amount of antifungal drugs are used for treatment at this time, the patient's own immune function may be affected, causing the beneficial bacteria in the intestines to be killed by antibacterial drugs, which is not conducive to the stability of the intestinal flora. Microecological regulators are widely used in patients with diarrhea. They can supplement the number of normal intestinal flora and stimulate the growth and reproduction of normal flora. When the number of normal flora occupies a large number in the intestine, they can inhibit the excessive reproduction and growth of pathogenic bacteria and effectively inhibit the long-term diarrhea caused by the continuous reproduction of pathogenic bacteria. Combined with the use of targeted antifungal drugs, the corresponding therapeutic effect can be achieved.

[0004] Therefore, there is a need in the art for a product that can simply and quickly detect the above-mentioned pathogens with high sensitivity and good specificity, which is conducive to providing timely clinical treatment strategies. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides a composition for detecting intestinal fungal infection and monitoring flora restoration, comprising upstream and downstream primers and probes for detecting at least 4 of the following related pathogens:

[0006] Upstream and downstream primers and probes for detecting Bifidobacterium as shown in SEQ ID NOs: 1 to 3;

[0007] Upstream and downstream primers and probes for detecting lactobacillus as shown in SEQ ID NOs: 4 to 6;

[0008] Upstream and downstream primers and probes for detecting Candida tropicalis as shown in SEQ ID NOs: 7 to 9;

[0009] Upstream and downstream primers and probes for detecting Fusarium fungi as shown in SEQ ID NOs: 10 to 12;

[0010] The upstream and downstream primers and probes for detecting Candida albicans as shown in SEQ ID NOs: 13 to 15; or

[0011] The upstream and downstream primers and probes for detecting Mucor are shown in SEQ ID NOs: 16-18.

[0012] The joint detection composition provided by the present invention mainly uses a multiplex fluorescent PCR analysis method to detect relevant pathogens, thereby simultaneously realizing the detection and differentiation of at least four pathogens among bifidobacteria, lactobacilli, tropical Candida, Fusarium, Candida albicans, and Mucor in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. At the same time, the composition of the present invention has a higher detection sensitivity of 200 copies / mL, and the detection is more accurate, which is conducive to providing preventive policies in a timely manner.

[0013] The present invention provides a composition for detecting intestinal fungal infection and monitoring flora repair, comprising:

[0014] Upstream and downstream primers and probes for detecting Bifidobacterium as shown in SEQ ID NOs: 1 to 3;

[0015] Upstream and downstream primers and probes for detecting lactobacillus as shown in SEQ ID NOs: 4 to 6;

[0016] Upstream and downstream primers and probes for detecting Candida tropicalis as shown in SEQ ID NOs: 7 to 9;

[0017] Upstream and downstream primers and probes for detecting Fusarium fungi as shown in SEQ ID NOs: 10 to 12;

[0018] Upstream and downstream primers and probes for detecting Candida albicans as shown in SEQ ID NOs: 13 to 15; and

[0019] The upstream and downstream primers and probes for detecting Mucor are shown in SEQ ID NOs: 16-18.

[0020] Furthermore, the composition includes upstream and downstream primers and probes for detecting the internal standard.

[0021] In some specific embodiments, the internal standard is a human internal standard gene.

[0022] In a specific embodiment, the internal standard is a human housekeeping gene.

[0023] Furthermore, the fluorescent groups of different probes in the composition of the present invention are different from each other and do not interfere with each other.

[0024] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX, CY5 and CY5.5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0025] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe sets. In the present invention, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0026] The composition of the present invention can be combined into any combination of the corresponding six targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0027] For example, it may include any 5 of the above 6 sets of primers and probes, it may include any 4 of the above 6 sets of primers and probes, it may include any 3 of the above 6 sets of primers and probes, it may include any 2 of the above 6 sets of primers and probes, or it may include any 1 of the above 6 sets of primers and probes.

[0028] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0029] In a specific embodiment, the fluorescent reporter group of the probe for Bifidobacterium is ATTO 425; the fluorescent reporter group of the probe for Lactobacillus is FAM; the fluorescent reporter group of the probe for Candida tropicalis is HEX; the fluorescent reporter group of the probe for Fusarium is ROX; the fluorescent reporter group of the probe for Candida albicans is CY5; and the fluorescent reporter group of the probe for Mucor is Quasar 705.

[0030] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0031] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, Dabcyl BHQ-0, BHQ1, BHQ2 or BHQ3.

[0032] Furthermore, the dosage of a single primer in the composition is 0.01 to 0.5 μM; the dosage of a single probe in the composition is 0.005 to 0.2 μM.

[0033] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0034] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0035] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0036] 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 intestinal fungal infection and monitoring flora repair, wherein the types of related pathogens are bifidobacteria, lactobacilli, tropical Candida, Fusarium, Candida albicans, and Mucor.

[0037] In a third aspect, the present invention provides a kit for detecting intestinal fungal infection and monitoring flora restoration, the kit comprising the composition of the present invention as described above.

[0038] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0039] In a specific embodiment, the negative control is at least one of DEPC H2O, physiological saline, and internal standard genes. The positive control is at least one of the plasmid or fragment genes of Bifidobacterium, Lactobacillus, Candida tropicalis, Fusarium, Candida albicans, and Mucor.

[0040] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0041] Furthermore, the amplification reagent includes dNTP(U)s, PCR buffer, DNA polymerase, and Mg 2+ At least one of .

[0042] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0043] Furthermore, the concentration of the DNA polymerase is 1 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0044] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, Mg 2+ , dNTP(U)s, primers, probes, UNG enzyme and PCR buffer.

[0045] PCR buffer is composed of Tris-HCl, MgCl2, KCl, Triton X-100, etc. The total volume in a single PCR reaction tube is generally 20 μl to 200 μl.

[0046] In a fourth aspect, a method for detecting intestinal fungal infection and monitoring flora restoration is provided, the method comprising the following steps:

[0047] 1) Extracting nucleic acid from the sample to be tested;

[0048] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0049] 3) Obtain and analyze the results.

[0050] In the present invention, the sample used for detection can be gastrointestinal secretions, feces, serum, blood, etc., but is not limited thereto.

[0051] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0052] UNG enzyme reaction, temperature is 50-65°C, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99°C, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95°C, time is 5-30 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0053] In a specific embodiment, a method for detecting intestinal fungal infection and monitoring flora restoration for non-diagnostic purposes is provided, the method comprising the following steps:

[0054] 1) Extracting nucleic acid from the sample to be tested;

[0055] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0056] 3) Obtain and analyze the results.

[0057] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0058] UNG enzyme reaction, temperature is 50-65°C, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99°C, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95°C, time is 5-30 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0059] In a specific embodiment, a composition is provided for preparing a reagent for detecting intestinal fungal infection and monitoring flora repair, wherein the use detection comprises the following steps:

[0060] 1) Extracting nucleic acid from the sample to be tested;

[0061] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0062] 3) Obtain and analyze the results.

[0063] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0064] UNG enzyme reaction, temperature is 50-65°C, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99°C, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95°C, time is 5-30 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0065] 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 and drinking water sources have the above pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figures 1 to 3 A graph showing the test results of different sample combinations of the composition of the present invention;

[0067] Figure 4 It is the sensitivity result diagram of the composition of the present invention;

[0068] Figure 5 It is a specific result diagram of the composition of the present invention;

[0069] Figure 6 It is a test result diagram of the comparative example composition of the present invention. DETAILED DESCRIPTION

[0070] 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.

[0071] Example 1: Primers and probes used in the present invention

[0072] Table 1

[0073]

[0074] The fluorescent reporter group of the probe for Bifidobacterium is ATTO 425; the fluorescent reporter group of the probe for Lactobacillus is FAM; the fluorescent reporter group of the probe for Candida tropicalis is HEX; the fluorescent reporter group of the probe for Fusarium is ROX; the fluorescent reporter group of the probe for Candida albicans is CY5; and the fluorescent reporter group of the probe for Mucor is Quasar705.

[0075] Example 2: Method for detecting intestinal fungal infection and monitoring flora restoration

[0076] The sample detected by the present invention is feces. The real-time fluorescence PCR reaction solution is configured according to the following Table 2:

[0077] Table 2

[0078]

[0079]

[0080] Enzyme preparation:

[0081] The enzyme is directly composed of 2 μL Anstart Taq DNA Polymerase enzyme (15 U / μL); Anstart Taq DNA Polymerase (15 U / μL) is purchased from Guangdong Feipeng Biological Co., Ltd.

[0082] Reagent preparation:

[0083] Take the corresponding amount of PCR reaction solution and enzyme mixture (PCR reaction solution 38 μL / person + enzyme 2 μL / person) in proportion, mix thoroughly to form PCR mixture, centrifuge at 2000 rpm for 10 seconds and set aside.

[0084] Sample handling and loading:

[0085] Take 300 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. according to its instructions to extract nucleic acid.

[0086] 10 μL of the above-treated samples, negative controls and positive controls were pipetted and added to reaction tubes respectively. 40 μL of PCR mixture was added to each reaction tube and the tube cap was covered.

[0087] PCR amplification:

[0088] On the fully automatic medical PCR analysis system, PCR amplification is performed according to a certain temperature and time setting program. The PCR amplification program is set as shown in Table 3 below:

[0089] Table 3

[0090]

[0091] If ATTO 425, FAM, HEX, CY5, ROX and Quasar705 channels have obvious S-shaped amplification curves and Ct values ​​≤38, they are judged as positive; if ATTO 425, FAM, HEX, ROX and Quasar705 channels have no amplification curves (No Ct) or Ct values ​​>38, and CY5 ≤38, they are judged as negative; if ATTO 425, FAM, HEX, ROX and Quasar705 channels have no amplification curves (No Ct) or Ct values ​​>38, and CY5 >38, the results are unreliable and need to be retested; the details are shown in Table 4.

[0092] Table 4. Interpretation of test results

[0093]

[0094] Example 3: Test results of the test samples of the composition of the present invention

[0095] The primers and probes shown in Example 1 were verified according to the method of Example 2 on samples of Bifidobacterium, Lactobacillus, Candida tropicalis, Fusarium, Candida albicans, and Mucor. The results showed that the composition of the present invention can detect and distinguish any combination of the above-mentioned related pathogens. For example, the composition of the present invention detects:

[0096] Bifidobacterium, Lactobacillus, Candida tropicalis, Candida albicans ( Figure 1 );

[0097] Bifidobacterium, Lactobacillus, Candida tropicalis, Fusarium, Mucor ( Figure 2 );

[0098] Bifidobacterium, Lactobacillus, Candida tropicalis, Fusarium, Candida albicans, Mucor ( Figure 3 ).

[0099] These figures prove that the present invention is a 6-plex PCR that can amplify the above 6 targets at one time and can be distinguished using a 6-channel PCR instrument. It also proves that any corresponding pair of combinations of the composition of the present invention can detect any corresponding combination of pathogens.

[0100] Example 4: Sensitivity of the composition of the present invention

[0101] Using the composition in Example 1 of the present invention, LOD (sensitivity) detection was performed on each target to simulate clinical samples and multiplex PCR detection was performed on a fluorescent quantitative PCR instrument. The results are as follows: Figure 4As shown, it is shown that each channel can still accurately detect samples as low as 200 copies / mL, indicating that the sensitivity of the composition of the present invention is 200 copies / mL.

[0102] Example 5. Specificity of the composition of the present invention

[0103] The composition of the present invention has no cross reaction with common pathogens and other pathogens with similar infection symptoms (Enterococcus faecalis, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Yersinia intestinalis, Shigella, Campylobacter, Escherichia coli O157, Clostridium difficile, Clostridium perfringens, Helicobacter pylori, enterovirus 71, astrovirus, norovirus, rotavirus, enteroadenovirus). The results are as follows Figure 5 As shown, it is shown that the composition of the present invention has good specificity.

[0104] Example 6: Anti-interference performance of the composition of the present invention

[0105] The composition of the embodiment was analyzed for interferences by conventional methods. The experimental results showed that potential PCR inhibitors / interfering substances such as dexamethasone (50 μg / mL), gentamicin (50 μg / mL), vancomycin (50 μg / mL), cefmenoxime hydrochloride (50 μg / mL), ciprofloxacin (50 μg / mL), co-trimoxazole (50 μg / mL), clindamycin (50 μg / mL), heme (10 μg / mL), hemoglobin (10%), etc. at certain concentrations had no obvious effect on the kit. The amplification results of the PCR reaction solution in the presence of infectious substances are specifically shown in Table 5 below.

[0106] Table 5

[0107]

[0108]

[0109] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0110] 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.

[0111] Therefore, the inventors also designed some other primers and probes to form a different detection system 2 (the sequences are shown in Table 6, and the primer and probe sequences in the system are replaced), which is also used to detect the above pathogens. Figure 6 As shown in the figure, it can be seen that the detection performance of the six pathogen targets is reduced, the Ct value is delayed, and the fluorescence increment is reduced.

[0112] Table 6

[0113]

[0114]

Claims

1. A composition for detecting intestinal fungal infection and monitoring flora restoration, comprising upstream and downstream primers and probes for detecting at least 4 of the following related pathogens: Upstream and downstream primers and probes for detecting Bifidobacterium as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting lactobacillus as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Candida tropicalis as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Fusarium fungi as shown in SEQ ID NOs: 10 to 12; The upstream and downstream primers and probes for detecting Candida albicans as shown in SEQ ID NOs: 13 to 15; or The upstream and downstream primers and probes for detecting Mucor are shown in SEQ ID NOs: 16-18.

2. The composition according to claim 1, characterized in that The composition comprises: Upstream and downstream primers and probes for detecting Bifidobacterium as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting lactobacillus as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Candida tropicalis as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Fusarium fungi as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Candida albicans as shown in SEQ ID NOs: 13 to 16; and The upstream and downstream primers and probes for detecting Mucor are shown in SEQ ID NOs: 16-18.

3. The composition according to claim 1, characterized in that The composition further comprises: Detect upstream and downstream primers and probes of internal standard.

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

5. The composition according to claim 4, characterized in that The fluorescent reporter group of the probe for Bifidobacterium is ATTO 425; the fluorescent reporter group of the probe for Lactobacillus is FAM; the fluorescent reporter group of the probe for Candida tropicalis is HEX; the fluorescent reporter group of the probe for Fusarium is ROX; the fluorescent reporter group of the probe for Candida albicans is CY5; and the fluorescent reporter group of the probe for Mucor is Quasar 705.

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

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a kit for detecting intestinal fungal infection and monitoring flora repair, wherein: The type of the relevant pathogen is one or more of Bifidobacterium, Lactobacillus, Candida tropicalis, Fusarium, Candida albicans or Mucor.

8. A kit for detecting intestinal fungal infection and monitoring flora restoration, the kit comprising the composition according to any one of claims 1 to 6.

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

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

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