Composition for detecting intestinal microorganisms

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 detection and distinguishing of at least four of clostridium praerzii, eubacterium rectum, streptococcus salivarius, clostridium tenella, prevotella and escherichia coli are simultaneously realized in a single-tube reaction system. Meanwhile, the detection sensitivity of the composition is higher and reaches 500 copies / mL, and the detection is more accurate.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and in particular, relates to a composition for detecting and distinguishing intestinal microorganisms, including Faecalibacterium prausnitzii, Eubacterium rectum, Streptococcus salivarius, Faecalibacterium flocculatum, Prevotella spp., and Escherichia coli. Background Art

[0002] The relationship between intestinal microorganisms and human health has attracted increasing attention from the scientific community. They are not only involved in the digestion and absorption of food, but also play an important role in immune regulation, metabolic balance, nervous system function and many other aspects.

[0003] Intestinal microorganisms are generally believed to have the following functions: Faecalibacterium prausnitzii (can produce anti-inflammatory factors and protect intestinal health); Eubacterium rectum (produces short-chain fatty acids, which play an important role in regulating intestinal function); Streptococcus salivarius (a rare probiotic strain that can improve allergic constitution and prevent recurrent allergies. Its function is to adjust allergic constitution. Inhibit the growth of pathogens and exert immune effects); Streptococcus (common intestinal colonizing bacteria, which can reduce safety risks, but when the body's immunity is reduced, it can cause inflammatory diseases such as meningitis and pneumonia); Prevotella (can help break down proteins and carbohydrates, and can also act as a conditional pathogen, causing periodontal and dental inflammation, intestinal inflammation, rheumatoid arthritis, bacterial vaginitis, etc.); Escherichia coli (is a normal resident bacteria in the intestines of humans and animals, and is also a conditional pathogen. Under certain conditions, it can cause gastrointestinal infections in humans and various animals or various local tissue and organ infections such as the urethra).

[0004] Understanding the abundance of intestinal microorganisms can help individuals take targeted health management measures, such as adjusting dietary structure, increasing the intake of probiotics, avoiding the abuse of antibiotics, etc., to maintain the balance and stability of the intestinal microecology.

[0005] 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 medical strategies. Summary of the invention

[0006] In view of this, in a first aspect, the present invention provides a composition for detecting intestinal microorganisms, comprising upstream and downstream primers and probes for detecting at least 4 of the following related pathogens:

[0007] Upstream and downstream primers and probes for detecting Faecalibacterium prausnitzii as shown in SEQ ID NOs: 1 to 3;

[0008] Upstream and downstream primers and probes for detecting Eubacterium rectum as shown in SEQ ID NOs: 4 to 6;

[0009] Upstream and downstream primers and probes for detecting Streptococcus salivarius as shown in SEQ ID NOs: 7 to 9;

[0010] Upstream and downstream primers and probes for detecting Clostridium truncatum as shown in SEQ ID NOs: 10 to 12;

[0011] The upstream and downstream primers and probes for detecting Prevotella as shown in SEQ ID NOs: 13 to 15; or

[0012] The upstream and downstream primers and probes for detecting Escherichia coli are shown in SEQ ID NOs: 16-18.

[0013] 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 Faecalibacterium prausnitzii, Eubacterium rectum, Streptococcus salivarius, Clostridium perfringens, Prevotella, and Escherichia coli in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. At the same time, the composition of the present invention has a higher detection sensitivity of 500 copies / mL, and its detection is more accurate than the melting curve, which is conducive to providing medical strategies in a timely manner.

[0014] The present invention provides a composition for detecting intestinal microorganisms, comprising:

[0015] Upstream and downstream primers and probes for detecting Faecalibacterium prausnitzii as shown in SEQ ID NOs: 1 to 3;

[0016] Upstream and downstream primers and probes for detecting Eubacterium rectum as shown in SEQ ID NOs: 4 to 6;

[0017] Upstream and downstream primers and probes for detecting Streptococcus salivarius as shown in SEQ ID NOs: 7 to 9;

[0018] Upstream and downstream primers and probes for detecting Clostridium truncatum as shown in SEQ ID NOs: 10 to 12;

[0019] Upstream and downstream primers and probes for detecting Prevotella as shown in SEQ ID NOs: 13 to 15; and

[0020] The upstream and downstream primers and probes for detecting Escherichia coli are shown in SEQ ID NOs: 16-18.

[0021] Furthermore, the composition includes upstream and downstream primers and probes for detecting the internal standard.

[0022] In some specific embodiments, the internal standard is a human internal standard gene.

[0023] In a specific embodiment, the internal standard is a human housekeeping gene.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0030] In a specific embodiment, the fluorescent reporter group of the Faecalibacterium prausnitzii probe is ATTO425; the fluorescent reporter group of the Eubacterium rectum probe is FAM; the fluorescent reporter group of the Streptococcus salivarius probe is HEX; the fluorescent reporter group of the Clostridium tenuis probe is ROX; the fluorescent reporter group of the Prevotella probe is CY5; and the fluorescent reporter group of the Escherichia coli probe is Quasar 705.

[0031] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0032] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, Dabcyl BHQ-0, BHQ1, BHQ2 or BHQ3.

[0033] 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.

[0034] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0035] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0036] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0037] In a second aspect, the present invention provides a use of the above-mentioned composition of the present invention in the preparation of a kit for detecting intestinal microorganisms, wherein the type of the relevant pathogen is one or more of Faecalibacterium prausnitzii, Eubacterium rectum, Streptococcus salivarius, Clostridium perfringens, Prevotella, and Escherichia coli.

[0038] In a third aspect, the present invention provides a kit for detecting intestinal microorganisms, wherein the kit comprises the composition of the present invention as described above.

[0039] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0040] In a specific embodiment, the negative control is at least one of DEPC H2O, physiological saline, and an internal standard gene. The positive control is at least one of a plasmid or a fragment gene of Faecalibacterium prausnitzii, Eubacterium rectum, Streptococcus salivarius, Clostridium licheniformis, Prevotella, and Escherichia coli.

[0041] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0042] Furthermore, the amplification reagent includes dNTP(U)s, PCR buffer, DNA polymerase, and Mg 2+ At least one of .

[0043] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0044] Furthermore, the concentration of the DNA polymerase is 1 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0045] 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.

[0046] 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.

[0047] In a fourth aspect, a method for detecting intestinal microorganisms is provided, the method comprising the following steps:

[0048] 1) Extracting nucleic acid from the sample to be tested;

[0049] 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);

[0050] 3) Obtain and analyze the results.

[0051] In the present invention, the sample used for detection may be feces, intestinal secretions, etc., but is not limited thereto.

[0052] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0053] 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.

[0054] In a specific embodiment, a method for detecting intestinal microorganisms for non-diagnostic purposes is provided, the method comprising the following steps:

[0055] 1) Extracting nucleic acid from the sample to be tested;

[0056] 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);

[0057] 3) Obtain and analyze the results.

[0058] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0059] 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.

[0060] In a specific embodiment, a composition is provided for use in preparing a reagent for detecting intestinal microorganisms, wherein the use detects the following steps:

[0061] 1) Extracting nucleic acid from the sample to be tested;

[0062] 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);

[0063] 3) Obtain and analyze the results.

[0064] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0065] 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.

[0066] In this context, the term "non-diagnostic purpose" means that the method is not intended to obtain information on whether an individual is infected with the above-mentioned pathogens and suffers from the infection. For example, the method can detect whether the environment and culture have the above-mentioned pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figures 1 to 3 A graph showing the test results of different sample combinations of the composition of the present invention;

[0068] Figure 4 It is a specific result diagram of the composition of the present invention;

[0069] Figures 5-6 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] Among them, the fluorescent reporter group of the Faecalibacterium prausnitzii probe is ATTO425; the fluorescent reporter group of the Eubacterium rectale probe is FAM; the fluorescent reporter group of the Streptococcus salivarius probe is HEX; the fluorescent reporter group of the Clostridium tenuis probe is ROX; the fluorescent reporter group of the Prevotella probe is CY5; and the fluorescent reporter group of the Escherichia coli probe is Quasar 705.

[0075] Example 2: Method for detecting intestinal microorganisms

[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] Reagent preparation:

[0080] Take the corresponding amount of PCR reaction solution and enzyme mixture (PCR reaction solution 38 μL / person + enzyme mixture 2 μL / person) in proportion, and mix them thoroughly to form a PCR mixture.

[0081] Sample handling and loading:

[0082] 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.

[0083] 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.

[0084] PCR amplification:

[0085] 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:

[0086] Table 3

[0087]

[0088]

[0089] If ATTO 425, FAM, HEX, CY5, ROX and Quasar705 channels have obvious S-shaped amplification curves and Ct values ​​≤ 40, it is judged as positive; if ATTO 425, FAM, HEX, ROX and Quasar705 channels have no amplification curve (No Ct) or Ct values ​​> 40, and CY5 ≤ 40, it is judged as negative; if ATTO 425, FAM, HEX, ROX and Quasar705 channels have no amplification curve (No Ct) or Ct values ​​> 40, and CY5 > 40, the result is unreliable and needs to be retested. The details are shown in Table 4.

[0090] Table 4. Interpretation of test results

[0091]

[0092] Example 3: Test results of the test samples of the composition of the present invention

[0093] The primers and probes shown in Example 1 were verified according to the method of Example 2 on samples of Faecalibacterium prausnitzii, Eubacterium rectum, Streptococcus salivarius, Clostridium truncatum, Prevotella, and Escherichia coli. 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:

[0094] Faecalibacterium prausnitzii, Prevotella, Streptococcus salivarius, Escherichia coli ( Figure 1 );

[0095] Faecalibacterium prausnitzii, Eubacterium rectale, Streptococcus salivarius, Prevotella, Escherichia coli ( Figure 2 );

[0096] Faecalibacterium prausnitzii, Eubacterium rectum, Streptococcus salivarius, Clostridium perfringens, Prevotella, Escherichia coli ( Figure 3 ).

[0097] 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 combination of the composition of the present invention can detect any corresponding combination of pathogens.

[0098] Example 4: Sensitivity of the composition of the present invention

[0099] Using the composition in Example 1 of the present invention, LOD (sensitivity) detection was performed on each target to simulate clinical samples and multiple PCR detection was performed on a fluorescent quantitative PCR instrument. The results are shown in Table 5, indicating that each channel can still accurately detect samples as low as 500 copies / mL, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0100] Table 5

[0101]

[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 4 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 kit of Example 2 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 6 below.

[0106] Table 6

[0107]

[0108] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0109] 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.

[0110] Therefore, the inventors also designed some other primers and probes to form a different detection system 2 (the sequence is shown in Table 7, replacing SEQ ID NO.17 with SEQ ID NO.19), which has a better effect when only detecting Escherichia coli (such as Figure 5 ), but it is also not effective in detecting the other pathogens mentioned above. Figure 6 As shown in the figure, it can be seen from the amplification curve that the detection performance of Clostridium perfringens and Escherichia coli is reduced, and the Ct value of the same template is significantly delayed.

[0111] Table 7

[0112]

Claims

1. A composition for detecting intestinal microorganisms, 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 Faecalibacterium prausnitzii as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Eubacterium rectum as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Streptococcus salivarius as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Clostridium truncatum as shown in SEQ ID NOs: 10 to 12; The upstream and downstream primers and probes for detecting Prevotella as shown in SEQ ID NOs: 13 to 15; or The upstream and downstream primers and probes for detecting Escherichia coli 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 Faecalibacterium prausnitzii as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Eubacterium rectum as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Streptococcus salivarius as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Clostridium truncatum as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Prevotella as shown in SEQ ID NOs: 13 to 16; and The upstream and downstream primers and probes for detecting Escherichia coli 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 Faecalibacterium prausnitzii probe is ATTO425; the fluorescent reporter group of the Eubacterium rectum probe is FAM; the fluorescent reporter group of the Streptococcus salivarius probe is HEX; the fluorescent reporter group of the Clostridium tenuis probe is ROX; the fluorescent reporter group of the Prevotella probe is CY5; and the fluorescent reporter group of the Escherichia coli probe 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 preparing a kit for detecting intestinal microorganisms, wherein: The type of the relevant pathogen is one or more of Faecalibacterium prausnitzii, Eubacterium rectum, Streptococcus salivarius, Clostridium perfringens, Prevotella, or Escherichia coli.

8. A kit for detecting intestinal microorganisms, 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 microorganisms, 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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