Composition and kit for detecting diarrhea-related pathogens and application of composition and kit

Abstract

The invention belongs to the field of molecular biological detection, and provides a combined detection composition which is mainly used for detecting diarrhea-related pathogen viruses by using a multiple fluorescent PCR (Polymerase Chain Reaction) analysis method. Therefore, at least four of human cystoprotozoa, cyclosporidium, binuclear ameeba fragilis, cryptosporidium, giardia and entamoeba histolytica can be 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 in particular, relates to a composition and use for detecting and distinguishing diarrhea-related pathogens, such as Blastocystis hominis, Cyclospora, Dinucleate Entamoeba fragilis, Cryptosporidium, Giardia lamblia, and Entamoeba histolytica. Background Art

[0002] Diarrhea is a common multiple disease with many causes, with bacterial infection being the most common. At the same time, protozoan diarrhea is also relatively common. Protozoan diarrhea usually refers to parasitic diseases that parasitize in the intestines or adjacent organs, with diarrhea and related symptoms as the main clinical manifestations or at a certain stage. Diarrhea may be caused by parasites (protozoa) damaging the intestinal wall tissue. Some of these worm species mainly damage the intestinal microvilli and villi, while others cause inflammation and ulcers of the intestinal wall, with obvious cell infiltration reactions around them. The protozoa that cause diarrhea mainly include: Trichuris trichuris, Entamoeba histolytica, Schistosoma japonicum, Cryptosporidium, Dienucleate Entamoeba fragilis, Giardia, Trichomonas humanus, Blastocystis humanus, Cyclospora and Balantidium coli, etc. Among them, Cryptosporidium, Entamoeba histolytica, Giardia, Blastocystis humanus, Cyclospora and Dienucleate Entamoeba fragilis can all parasitize in the human intestine and cause diarrhea in patients.

[0003] Entamoeba histolytica Schaudinn is a member of the genus Entamoeba of the family Entamoeba, which causes amebiasis.

[0004] Giardia lamblia Stiles, also known as Giardia, parasitizes the small intestine and gallbladder of the human body, mainly in the duodenum, and can cause symptoms such as abdominal pain, diarrhea and malabsorption, leading to giardiasis. It is one of the common parasites that infect the human intestinal tract.

[0005] Cryptosporidium is a parasitic single-cell protozoan belonging to the kingdom Protozoa. Cryptosporidium parvum (Cp) is the main pathogen that infects humans and most vertebrates. Cryptosporidium parvum is an important opportunistic pathogen distributed worldwide, which can cause cryptosporidiosis, a zoonotic disease.

[0006] Blastocystis hominis is one of the most common intestinal parasitic protozoa. It is a pathogenic protozoan that parasitizes the intestines of higher primates and humans. The main transmission route is fecal-oral transmission. Ingestion of food or water contaminated by Blastocystis hominis can cause infection with this parasite.

[0007] Cyclospora cayetanensis is a zoonotic parasite that mainly parasitizes in intestinal epithelial cells. It has a typical single-host life cycle and is widely present in a variety of vertebrates. It is a protozoan disease with diarrhea as the main clinical manifestation.

[0008] Dientamoeba fragilis is an amoeba-type flagellate that can parasitize in the human intestine or even outside the intestine. Under certain conditions, it can invade the intestinal wall tissue or other organ tissues. The main symptoms are diarrhea, abdominal pain, blood or mucus in the stool, nausea, vomiting, etc.

[0009] 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

[0010] 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 four of the following diarrhea-related pathogens:

[0011] Upstream and downstream primers and probes for detecting Blastocystis hominis as shown in SEQ ID NOs: 1 to 3;

[0012] Upstream and downstream primers and probes for detecting Cyclospora as shown in SEQ ID NOs: 4 to 6;

[0013] Upstream and downstream primers and probes for detecting Dikaryotic Entamoeba fragilis as shown in SEQ ID NOs: 7 to 9;

[0014] Upstream and downstream primers and probes for detecting Cryptosporidium as shown in SEQ ID NOs: 10 to 12;

[0015] An upstream primer for detecting Giardia as shown in SEQ ID NO: 13, at least one of the downstream primers for detecting Giardia as shown in SEQ ID NO: 14 or 15, and a probe for detecting Giardia as shown in SEQ ID NO: 16; or

[0016] The upstream and downstream primers and probes for detecting Entamoeba histolytica are shown in SEQ ID NOs: 17-19.

[0017] The combined detection composition provided by the present invention mainly uses a multiplex fluorescent PCR analysis method to detect diarrhea-related pathogens, thereby simultaneously realizing the detection and differentiation of at least four pathogens of human Blastocystis, Cyclospora, Dinucleate Amoeba Fragilis, Cryptosporidium, Giardia, and Entamoeba histolytica 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.

[0018] The present invention provides a composition for detecting diarrhea-related pathogens, comprising:

[0019] Upstream and downstream primers and probes for detecting Blastocystis hominis as shown in SEQ ID NOs: 1 to 3;

[0020] Upstream and downstream primers and probes for detecting Cyclospora as shown in SEQ ID NOs: 4 to 6;

[0021] Upstream and downstream primers and probes for detecting Dikaryotic Entamoeba fragilis as shown in SEQ ID NOs: 7 to 9;

[0022] Upstream and downstream primers and probes for detecting Cryptosporidium as shown in SEQ ID NOs: 10 to 12;

[0023] An upstream primer for detecting Giardia as shown in SEQ ID NO: 13, at least one of the downstream primers for detecting Giardia as shown in SEQ ID NO: 14 or 15, and a probe for detecting Giardia as shown in SEQ ID NO: 16; or

[0024] The upstream and downstream primers and probes for detecting Entamoeba histolytica are shown in SEQ ID NOs: 17-19.

[0025] Furthermore, the composition includes downstream primers for detecting Giardia as shown in SEQ ID NOs: 14 and 15.

[0026] By designing two downstream primers for Giardia lamblia, missed detection of Giardia lamblia can be further avoided.

[0027] Furthermore, the present invention provides a composition for detecting diarrhea-related pathogens, comprising:

[0028] Upstream and downstream primers and probes for detecting Blastocystis hominis as shown in SEQ ID NOs: 1 to 3;

[0029] Upstream and downstream primers and probes for detecting Cyclospora as shown in SEQ ID NOs: 4 to 6;

[0030] Upstream and downstream primers and probes for detecting Dikaryotic Entamoeba fragilis as shown in SEQ ID NOs: 7 to 9;

[0031] Upstream and downstream primers and probes for detecting Cryptosporidium as shown in SEQ ID NOs: 10 to 12;

[0032] Upstream and downstream primers and probes for detecting Giardia as shown in SEQ ID NOs: 13 to 16; and

[0033] The upstream and downstream primers and probes for detecting Entamoeba histolytica are shown in SEQ ID NOs: 17-19.

[0034] Furthermore, the composition includes upstream and downstream primers and probes for detecting the internal standard.

[0035] In some specific embodiments, the internal standard is a human internal standard gene.

[0036] In a specific embodiment, the internal standard is a human housekeeping gene.

[0037] 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.

[0038] 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.

[0039] Further, in some embodiments, the composition of the present invention may include one or more pairs of the above primer and probe pairs. In the present invention, "pair" refers to an upstream primer, a downstream primer and a probe that match each other to detect a target.

[0040] 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.

[0041] For example, it may include any 5 pairs of the above 6 pairs of primers and probes, it may include any 4 pairs of the above 6 pairs of primers and probes, it may include any 3 pairs of the above 6 pairs of primers and probes, it may include any 2 pairs of the above 6 pairs of primers and probes, or it may include any 1 pair of the above 6 pairs of primers and probes.

[0042] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0043] In a specific embodiment, the fluorescent reporter group of the probe for Entamoeba histolytica is ATTO 425; the fluorescent reporter group of the probe for Giardia lamblia is FAM; the fluorescent reporter group of the probe for Cryptosporidium is HEX; the fluorescent reporter group of the probe for Blastocystis hominis is ROX; the fluorescent reporter group of the probe for Cyclospora fragilis is CY5; and the fluorescent reporter group of the probe for Dinucleate Entamoeba fragilis is Quasar 705.

[0044] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0045] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, Dabcyl BHQ-0, BHQ1, BHQ2 or BHQ3.

[0046] 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.

[0047] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0048] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0049] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0050] 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 diarrhea-related pathogens, wherein the types of the diarrhea-related pathogens are Blastocystis hominis, Cyclospora, Dinucleate Entamoeba fragilis, Cryptosporidium, Giardia lamblia, and Entamoeba histolytica.

[0051] 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.

[0052] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0053] 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 the plasmid or fragment gene of Blastocystis hominis, Cyclospora, Dinucleate Entamoeba fragilis, Cryptosporidium, Giardia, and Entamoeba histolytica.

[0054] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0055] Furthermore, the amplification reagent includes dNTP(U)s, PCR buffer, reverse transcriptase, DNA polymerase, and Mg 2+ At least one of .

[0056] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0057] Furthermore, the concentration of the DNA polymerase is 1 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0058] 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.

[0059] 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.

[0060] In a fourth aspect, a method for detecting diarrhea-related pathogens is provided, the method comprising 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] In the present invention, the sample used for detection can be gastrointestinal secretions, feces, serum, blood, etc., but is not limited thereto.

[0065] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0066] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95℃, time is 5-30 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0067] In a specific embodiment, a method for detecting diarrhea-associated pathogens for non-diagnostic purposes is provided, the method comprising the following steps:

[0068] 1) Extracting nucleic acid from the sample to be tested;

[0069] 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);

[0070] 3) Obtain and analyze the results.

[0071] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0072] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95℃, time is 5-30 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0073] In a specific embodiment, a composition is provided for use in preparing a reagent for detecting diarrhea-related pathogens, wherein the use detects the following steps:

[0074] 1) Extracting nucleic acid from the sample to be tested;

[0075] 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);

[0076] 3) Obtain and analyze the results.

[0077] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0078] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95℃, time is 5-30 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figures 1 to 3 A graph showing the test results of different sample combinations of the composition of the present invention;

[0080] Figure 4 It is the sensitivity result diagram of the composition of the present invention;

[0081] Figure 5 It is a specific result diagram of the composition of the present invention;

[0082] Figure 6 It is a test result diagram of the comparative example composition of the present invention. DETAILED DESCRIPTION

[0083] 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.

[0084] Example 1: Primers and probes used in the present invention

[0085] Table 1

[0086]

[0087] The fluorescent reporter group of the probe for Entamoeba histolytica is ATTO 425; the fluorescent reporter group of the probe for Giardia lamblia is FAM; the fluorescent reporter group of the probe for Cryptosporidium is HEX; the fluorescent reporter group of the probe for Blastocystis hominis is ROX; the fluorescent reporter group of the probe for Cyclospora parvum is CY5; and the fluorescent reporter group of the probe for D-etermuclear Entamoeba fragilis is Quasar 705.

[0088] Example 2: Method for detecting diarrhea-related pathogens

[0089] The sample detected by the present invention is feces. The real-time fluorescence PCR reaction solution is configured according to the following Table 2:

[0090] Table 2

[0091] Components The volume of each reaction PCR Buffer 33.60μL Primer (50pmol / μL) 0.2μL / strip (11 strips), 0.4μL / strip (2 strips) Probe (50pmol / μL) 0.2μL / strip (1 strip) 0.1μL / strip (5 strips) dNTPs(100mM) 0.5μL <![CDATA[1mol / L MgCl2]]> 0.2μL

[0092] Preparation of enzyme mixture:

[0093] The enzyme mixture consists of UNG enzyme and H-Taq enzyme. H-Taq enzyme (5U / μL) and UNG enzyme (2U / μL) are mixed in a certain ratio (2μL H-Taq enzyme and 0.1μL UNG enzyme per person). The enzyme mixture is used in an amount of 2μL per person.

[0094] Reagent preparation:

[0095] Take the corresponding amount of PCR reaction solution and enzyme mixture (PCR reaction solution 38 μL / person + enzyme mixture 2 μL / person) in proportion, mix thoroughly to form PCR mixture, centrifuge at 2000 rpm for 10 seconds and set aside.

[0096] Sample handling and loading:

[0097] 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.

[0098] 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.

[0099] PCR amplification:

[0100] 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:

[0101] Table 3

[0102]

[0103]

[0104] 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.

[0105] Table 4. Interpretation of test results

[0106]

[0107] Example 3: Test results of the test samples of the composition of the present invention

[0108] The primers and probes shown in Example 1 were verified according to the method of Example 2 on samples of Blastocystis hominis, Cyclospora, Dinucleate Entamoeba fragilis, Cryptosporidium, Giardia lamblia, and Entamoeba histolytica. The results showed that the composition of the present invention can detect and distinguish any combination of the above diarrhea-related pathogens. For example, the composition of the present invention detects:

[0109] Blastocystis hominis, Cyclospora, Giardia lamblia, Dikaryotic amoeba ( Figure 1 );

[0110] Blastocystis hominis, Cyclospora, Cryptosporidium, Dikaryotic amoeba, Entamoeba histolytica ( Figure 2 );

[0111] Blastocystis hominis, Cyclospora, Dinucleate Entamoeba fragilis, Cryptosporidium, Giardia lamblia, Entamoeba histolytica ( Figure 3 ).

[0112] 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.

[0113] Example 4: Sensitivity of the composition of the present invention

[0114] 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 4 As shown in Table 5, 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.

[0115] Table 5

[0116]

[0117] Example 5. Specificity of the composition of the present invention

[0118] 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.

[0119] Example 6: Anti-interference and stability of the composition of the present invention

[0120] 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 6 below.

[0121] Table 6

[0122]

[0123] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0124] 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.

[0125] Therefore, the inventors also designed some other primers and probes to form a different detection system 2 (the sequences are shown in Table 7, with the primer and probe sequences of Blastocystis hominis 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 diarrheal protozoa targets is reduced, the Ct value is delayed, and the fluorescence increment is reduced.

[0126] Table 7

[0127]

[0128]

Claims

1. A composition for detecting diarrhea-related pathogens, comprising upstream and downstream primers and probes for detecting at least 4 of the following diarrhea-related pathogens: Upstream and downstream primers and probes for detecting Blastocystis hominis as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Cyclospora as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Dikaryotic Entamoeba fragilis as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Cryptosporidium as shown in SEQ ID NOs: 10 to 12; An upstream primer for detecting Giardia as shown in SEQ ID NO: 13, at least one of the downstream primers for detecting Giardia as shown in SEQ ID NO: 14 or 15, and a probe for detecting Giardia as shown in SEQ ID NO: 16; or The upstream and downstream primers and probes for detecting Entamoeba histolytica are shown in SEQ ID NOs: 17-19.

2. The composition according to claim 1, characterized in that The composition comprises: downstream primers for detecting Giardia as shown in SEQ ID NOs: 14 and 15.

3. The composition according to claim 1, characterized in that The composition comprises: Upstream and downstream primers and probes for detecting Blastocystis hominis as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Cyclospora as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Dikaryotic Entamoeba fragilis as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Cryptosporidium as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Giardia as shown in SEQ ID NOs: 13 to 16; and The upstream and downstream primers and probes for detecting Entamoeba histolytica are shown in SEQ ID NOs: 17-19.

4. The composition according to claim 3, characterized in that The fluorescent reporter group of the probe for Entamoeba histolytica is ATTO 425; the fluorescent reporter group of the probe for Giardia lamblia is FAM; the fluorescent reporter group of the probe for Cryptosporidium is HEX; the fluorescent reporter group of the probe for Blastocystis hominis is ROX; the fluorescent reporter group of the probe for Cyclospora parvum is CY5; and the fluorescent reporter group of the probe for D-etermuclear Entamoeba fragilis is Quasar 705.

5. The composition according to claim 4, characterized in that The 3' end of the probe also has a quencher group.

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 diarrhea-related pathogens, wherein: The type of diarrhea-related pathogen is one or more of Blastocystis hominis, Cyclospora, Dinucleate Entamoeba fragilis, Cryptosporidium, Giardia lamblia or Entamoeba histolytica.

8. A kit for detecting diarrhea-related pathogens, 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 diarrhea-related pathogens, 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.

Images