Composition for detecting diarrhea-related pathogens, kit and application

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of diarrhea-related pathogens, and more particularly relates to detection of at least four of morganella morganii, provingue, proteus mirabilis, enterobacter aerogenes and aeromonas caviridis. The composition disclosed by the invention is higher in detection sensitivity (up to 500 copies / mL), good in specificity and more accurate in detection, and can provide sufficient basis for rapid detection and exclusion of infection of different pathogens, shorten the judgment time of diarrhea inducements and accelerate implementation of countermeasures.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of diarrhea-related pathogens, and more specifically, to the detection of Morganella morganii, Providencia, Proteus mirabilis, Enterobacter aerogenes, and Aeromonas caviae. Background Art

[0002] Morganella morganii, also known as Morganella, is widely distributed in water, soil, and human and animal feces. It is a member of the normal intestinal flora of humans and animals, but it is also a conditional pathogen and a zoonosis pathogen. Morganella morganii is highly toxic and can not only cause serious infections, but can even lead to the death of many animals including humans. It can also cause food poisoning. After infection, clinical symptoms such as chills, chills, fever, and body aches often appear, and characteristic infection symptoms of decomposing proteins in the body to produce a foul odor.

[0003] Providencia belongs to the genus Providencia and the family Morganella. Providencia is currently divided into 7 complex groups and 18 genospecies, mainly including Providencia rettgeri, Providencia stuartii and Providencia alcalifaciens, which are prone to cause infection in patients with impaired immune function or long-term hospitalization. It is often transmitted through contact, such as hand contact by medical staff or contamination of medical equipment, leading to the occurrence of various infectious diseases such as diarrhea. In addition, Providencia can also become an important pathogen of traveler's diarrhea and other gastrointestinal infections, especially in areas with poor sanitary conditions or lax food safety control.

[0004] Proteus mirabilis is a Gram-negative bacterium of the family Enterobacteriaceae. As an important zoonotic pathogen, Proteus mirabilis is widely distributed in nature and can cause gastrointestinal, respiratory, and urinary tract infections in animals and humans. It is the main pathogen causing food poisoning, second only to Vibrio parahaemolyticus, Salmonella, and Staphylococcus aureus. Therefore, the prevention and treatment of Proteus mirabilis infection has important public health significance.

[0005] Enterobacter aerogenes is a conditional pathogen that mainly lives in the intestines of humans and animals. It is a Gram-negative facultative anaerobic bacillus belonging to the genus Enterobacter of the family Enterobacteriaceae. Although Enterobacter aerogenes is a normal strain in the human intestine, it can cause dysbiosis and become a pathogenic strain when the body's immunity is low. Clinically, Enterobacter aerogenes is common in causing disease, and drug resistance is also serious.

[0006] The number of reported cases of Aeromonas caviae has increased significantly, and it has become a common pathogen that affects humans, animals, and aquatic organisms. It is widely found in fresh water, sea water, sewage, soil, and silt, and can be pathogenic to animals under certain conditions. Currently, reports of infection with Aeromonas caviae mainly involve gastroenteritis caused by human infection.

[0007] Therefore, there is a need in the art for a product that can simply and quickly detect the above-mentioned pathogens, and has good sensitivity and specificity, and can detect and distinguish pathogenic microorganisms that cause diarrhea, so as to provide targeted strategies for the prevention and treatment of the above-mentioned diarrhea. Summary of the invention

[0008] In view of this, in a first aspect, the present invention provides a composition for detecting diarrhea-related pathogens, comprising upstream and downstream primers and probes for detecting at least 4 of the following pathogens:

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

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

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

[0012] The upstream primers, downstream primers and probe for detecting Enterobacter aerogenes as shown in SEQ ID NOs: 10 to 12; or

[0013] The upstream primers, downstream primers and probe for detecting Aeromonas caviae are shown in SEQ ID NOs: 13-15.

[0014] The combined detection composition provided by the present invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and differentiation of at least four of Morganella morganii, Providencia, Proteus mirabilis, Enterobacter aerogenes, and Aeromonas caviae in a single-tube reaction system, so as to provide a targeted strategy for the prevention and treatment of diarrhea. The composition of the present invention has a higher detection sensitivity of 500 copies / mL, good specificity, and more accurate detection, and can provide a relatively sufficient basis for rapid detection and exclusion of infection by different pathogens, shorten the judgment time of the cause of diarrhea, and accelerate the implementation of countermeasures.

[0015] Furthermore, the present invention provides a composition for detecting diarrhea-related pathogens, comprising:

[0016] Upstream primers, downstream primers and probes for detecting Morganella morganii as shown in SEQ ID NOs: 1 to 3;

[0017] Upstream primers, downstream primers and probes for detecting Providencia as shown in SEQ ID NOs: 4 to 6;

[0018] Upstream primers, downstream primers and probes for detecting Proteus mirabilis as shown in SEQ ID NOs: 7 to 9;

[0019] Upstream primers, downstream primers and probes for detecting Enterobacter aerogenes as shown in SEQ ID NOs: 10 to 12; and

[0020] The upstream primers, downstream primers and probe for detecting Aeromonas caviae are shown in SEQ ID NOs: 13-15.

[0021] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0022] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0023] In a specific embodiment, the internal standard is RNase P or GAPDH.

[0024] 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.

[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 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.

[0026] In some specific embodiments, the fluorescent reporter group of the probe for detecting Morganella morganii is FAM; the fluorescent reporter group of the probe for detecting Providencia is HEX; the fluorescent reporter group of the probe for detecting Proteus mirabilis is ROX; the fluorescent reporter group of the probe for detecting Enterobacter aerogenes is CY5; and the fluorescent reporter group of the probe for detecting Aeromonas caviae is QUASAR 705.

[0027] 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 primers, downstream primers and probes that match each other to detect a target.

[0028] The composition of the present invention can be combined into any combination of the corresponding five targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the upstream and downstream primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0029] For example, it may include any 4 of the above 5 sets of primers and probes, any 3 of the above 5 sets of primers and probes, any 2 of the above 5 sets of primers and probes, or any 1 of the above 5 sets of primers and probes.

[0030] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[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 BHQ1 or BHQ2.

[0033] In a specific embodiment, the quencher group at the 3' end of the probe is BHQ1.

[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 the use of the above-mentioned composition of the present invention in the preparation of a kit for detecting diarrhea-related pathogens, wherein the pathogen is one or more of Enterobacter aerogenes, Morganella morganii, Providencia, Proteus mirabilis, or Aeromonas caviae.

[0038] In a third aspect, the present invention provides a kit for detecting diarrhea-related pathogens, 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 and normal saline; the positive control is at least one of a fragment plasmid or a positive strain of Enterobacter aerogenes, Morganella morganii, Providencia, Proteus mirabilis, and Aeromonas caviae.

[0041] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0042] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG enzyme).

[0043] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0044] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UNG enzyme), DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0045] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0046] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UNG enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.

[0047] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0048] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0049] 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.

[0050] In a fourth aspect, a method for detecting diarrhea-related pathogens is provided, the method comprising 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] In the present invention, the sample used for detection may be feces or digestive tract secretions, etc., but is not limited thereto.

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

[0056] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0057] Furthermore, the method for detecting diarrhea-related pathogens is for non-diagnostic purposes.

[0058] In a specific embodiment, a composition is provided for use in preparing a reagent for detecting diarrhea-related pathogens, wherein the detection comprises the following steps:

[0059] 1) Extracting nucleic acid from the sample to be tested;

[0060] 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);

[0061] 3) Obtain and analyze the results.

[0062] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0063] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0064] In this context, the term "non-diagnostic purpose" refers to not being intended to obtain information on whether an individual is infected with the above pathogens and suffers from diarrhea. For example, the method can detect whether the above pathogens are present in the environment and in culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figures 1 to 3 The results of testing different samples for the composition of the present invention are shown in FIG.

[0066] Figure 4 This is a sensitivity result diagram of the composition of the present invention;

[0067] Figure 5 It is a specific result diagram of the composition of the present invention;

[0068] Figure 6 It is a single test result diagram of the composition of the present invention and the comparative example composition;

[0069] Figure 7 This is a diagram showing the joint test results 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] The primers and probes used in the present invention are shown in Table 1 below:

[0073] Table 1

[0074]

[0075] Among them, the fluorescent reporter group of the probe for detecting Morganella morganii is FAM; the fluorescent reporter group of the probe for detecting Providencia is HEX; the fluorescent reporter group of the probe for detecting Proteus mirabilis is ROX; the fluorescent reporter group of the probe for detecting Enterobacter aerogenes is CY5; and the fluorescent reporter group of the probe for detecting Aeromonas caviae is QUASAR 705.

[0076] Example 2: Method for detecting pathogens

[0077] Reagent preparation

[0078] 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 according to the proportion (PCR reaction solution 38 μL / person + enzyme mixture 2 μL / person), mix them thoroughly to form a PCR mixture, and centrifuge at 2000 rpm for 10 seconds before use.

[0079] The enzyme mixture consists of UNG enzyme and H-Taq enzyme. H-Taq enzyme (15U / μL) and UNG enzyme (1U / μL) are mixed in a certain ratio (0.9μL H-Taq enzyme and 0.1μL UNG enzyme per person).

[0080] The PCR reaction solution was prepared according to Table 2.

[0081] Table 2

[0082] Components The volume of each reaction PCR buffer* 19.20μL dNTPs(100mM) 0.50μL <![CDATA[1mol / L MgCl2]]> 0.20μL Primer (50pmol / μL) 0.2μL / strip Probe (50pmol / μL) 0.1μL / strip

[0083] Sample handling and loading

[0084] 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. according to its instructions to extract nucleic acid.

[0085] 5 μL of the above-treated samples, negative controls and positive controls were pipetted and added to the corresponding 0.2 mL PCR reaction tubes, 20 μL of PCR mixture was added to each tube, and the tubes were capped.

[0086] PCR amplification

[0087] PCR amplification was performed on a PCR instrument such as the SLAN-96P fully automatic medical PCR analysis system according to the temperature and time setting program shown in Table 3.

[0088] Table 3

[0089]

[0090] Result analysis and judgment

[0091] If the FAM, HEX, CY5, ROX and Quasar705 channels have obvious S-shaped amplification curves and the Ct value is ≤38, it is judged as positive; if the FAM, HEX, CY5, ROX and Quasar705 channels have no amplification curve (No Ct) or the Ct value is greater than 38, it is judged as negative; the details are shown in Table 4 below.

[0092] Table 4

[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 used to perform PCR detection on a mixed sample of Morganella morganii, Providencia, Proteus mirabilis, and Enterobacter aerogenes on a SLAN-96P real-time fluorescence quantitative PCR instrument according to the method of Example 2. 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] Providencia, Proteus mirabilis, Enterobacter aerogenes, Aeromonas caviae ( Figure 1 );

[0097] Morganella morganii, Proteus mirabilis, Enterobacter aerogenes, Aeromonas caviae ( Figure 2 );

[0098] Morganella morganii, Providencia, Proteus mirabilis, Enterobacter aerogenes, Aeromonas caviae ( Figure 3 ).

[0099] These figures can prove that the present invention is a 5-plex PCR that can amplify the above-mentioned 5 targets at one time and can be distinguished using a 5-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.

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

[0101] 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, and 500 copies / mL, respectively, and the four concentrations were tested. Among them, the test results of the plasmid samples of Morganella morganii, Providencia, Proteus mirabilis, Enterobacter aerogenes, and Aeromonas caviae in the concentration gradient are as follows: Figure 4 As shown in Table 5. The results show 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.

[0102] Table 5

[0103]

[0104] Example 5. Specificity of the composition of the present invention

[0105] The composition of the present invention has no cross reaction with diarrhea-related pathogens (Helicobacter pylori, enterovirus 71, astrovirus, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile, Clostridium perfringens, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Yersinia intestinalis, Shigella, Escherichia coli O157, Clostridium difficile, Vibrio fluvii and other diarrhea pathogens). Figure 5 As shown, it is shown that the composition of the present invention has good specificity.

[0106] Example 6: Anti-interference and stability of the composition of the present invention

[0107] The experimental results show that potential PCR inhibitors / interfering substances such as dexamethasone (50 μg / mL), gentamicin (50 μg / mL), vancomycin (50 μg / mL), ceftriaxone 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 a certain concentration have no obvious effect on the composition of the present invention. The results are shown in Table 6.

[0108] Table 6

[0109]

[0110]

[0111] Comparative Example 1: Test results of the test samples of the comparative example composition of the present invention

[0112] 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.

[0113] In the present invention, the combination of primers and probes is irreplaceable and replaceable. Other primers and probes are used to form different detection systems (replacing the primer and probe sequences of Aeromonas caviae, as shown in Table 7). Figure 6 It can be seen that the primer and probe set of Aeromonas caviae (combination 2) used in the comparative example is better than the primer and probe set of Aeromonas caviae (combination 1) in single target amplification. However, after combining it with the other 4 primer and probe sets of the composition of the present invention, it was found that ( Figure 7 ), the Ct value of the test is late, the detection performance of some targets is reduced, and the fluorescence increment decreases. Therefore, the overall detection effect is poor.

[0114] Table 7

[0115]

[0116]

Claims

1. A composition for detecting diarrhea-related pathogens, comprising upstream and downstream primers and probes for detecting at least 4 of the following pathogens: Upstream primers, downstream primers and probes for detecting Morganella morganii as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Providencia as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Proteus mirabilis as shown in SEQ ID NOs: 7 to 9; The upstream primers, downstream primers and probe for detecting Enterobacter aerogenes as shown in SEQ ID NOs: 10 to 12; or The upstream primers, downstream primers and probe for detecting Aeromonas caviae are shown in SEQ ID NOs: 13-15.

2. The composition according to claim 1, characterized in that The composition comprises: Upstream primers, downstream primers and probes for detecting Morganella morganii as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Providencia as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Proteus mirabilis as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting Enterobacter aerogenes as shown in SEQ ID NOs: 10 to 12; and The upstream primers, downstream primers and probe for detecting Aeromonas caviae are shown in SEQ ID NOs: 13-15.

3. The composition according to claim 2, characterized in that The fluorescent groups between the probes detecting different pathogens are different and do not interfere with each other.

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 diarrhea-related pathogens, wherein The pathogen is one or more of Enterobacter aerogenes, Morganella morganii, Providencia, Proteus mirabilis, or Aeromonas caviae.

7. A kit for detecting diarrhea-related pathogens, 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, a UNG enzyme, dNTP, dUTP, a PCR buffer, or Mg 2+ At least one of .

10. Use of a composition for preparing a reagent for detecting diarrhea-related pathogens, 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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