CN116970724A - Composition, kit, method and application for detecting intestinal tract-related pathogens - Google Patents
Composition, kit, method and application for detecting intestinal tract-related pathogens Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology detection, and particularly relates to detection of intestinal related pathogens, in particular to detection of Shigella, vibrio parahaemolyticus and salmonella typhi. The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that detection and differentiation of Shigella, vibrio parahaemolyticus and salmonella typhi are realized simultaneously in a single-tube reaction system. The composition has higher detection sensitivity reaching 1000 copies/mL, good specificity and more accurate detection.
Description
Technical Field
The invention belongs to the field of molecular biology detection, and particularly relates to detection of different types of intestinal related pathogens, in particular to detection of Shigella dysenteriae, vibrio parahaemolyticus and salmonella typhi.
Background
Diarrhea is one of the most common problems worldwide, is a common and frequently occurring disease that is harmful to human health, and enteropathogen infection is a major cause of diarrhea. As many as 20 million diarrhea cases are present annually worldwide, possibly associated with poor hygienic conditions. Food-borne diseases are a group of diseases that cause infection or poisoning of the human body due to ingestion of food with pathogenicity, and more than 250 food-borne pathogenic sources have been found, most of which are infectious pathogenic sources, mainly a variety of bacteria, viruses and parasites. Food-borne diseases caused by pathogenic bacteria have become a worldwide public health problem, and according to World Health Organization (WHO) statistics, food-borne diseases cause about 220 tens of thousands of deaths worldwide each year.
Vibrio parahaemolyticus (briopa haemolyticus) is widely found in estuary, marine and coastal environments. Vibrio parahaemolyticus is a main causative agent of acute gastroenteritis after eating improperly treated seafood by humans. In rare cases, vibrio parahaemolyticus may cause wound infection, ear infection or septicemia, and life may be endangered in severe cases.
Shigella (Plesiomonas Shigelloides) is an oxidase-positive, facultative anaerobic, gram-negative bacillus-free bacterium with an optimal growth temperature of 37 ℃, which is mainly found in fresh water, river water and sea water, and is particularly found in tropical and subtropical areas. The infection of the intestinal infection caused by Shigella is rare, the intestinal infection of human beings is mostly caused, clinical symptoms are diarrhea, abdominal pain, nausea, aversion to cold, headache, fever and vomiting, the diarrhea is mostly watery stool, the diarrhea is a small number of bloody stools, the incubation period is 24-50 hours, the course of the disease is generally 1-9 days, the infection course caused by a small number of virulent strains can reach 2 weeks-3 months, and children and immunodeficiency patients in the population are susceptible. In addition, infection in people with low immunity can cause cellulitis, osteomyelitis, meningitis, septicemia, etc. In the detection of food poisoning, shigella-like bacteria are often ignored because of the similarity of clinical symptoms of patients and other pathogenic bacteria, and diarrhea caused by unknown causes is diagnosed.
Salmonella is a wide variety, and so far a number of Salmonella serotypes 2600 have been co-found, of which more than 1500 can infect humans. Salmonella is classified into two major types, salmonella Typhi (Salmonella Typhi) and Salmonella non-Typhi ((Nontyphoidal Salmonella), according to the pathological changes occurring in the body after infection, salmonella Typhi mainly comprises typhoid, sendai, paratyphoid A, paratyphoid B and paratyphoid C, wherein the host range of Salmonella Typhi is narrow, and mainly infects humans and higher primates, wherein the clinical manifestations caused by Salmonella Typhi are relatively heavy, mainly intestinal heat, the latency period is long, the latency period can fluctuate for 4-33 days according to the number of infected bacteria, high heat (T >39 ℃) is the most common symptom, the duration of the symptom can be as long as 3 weeks, and other rare clinical complications such as meningitis and osteomyelitis can be caused.
Therefore, there is a need in the art for a product that can simply and quickly detect the different pathogens described above, so as to provide a basis for a clinician to diagnose and eliminate infection by the different pathogens more fully and quickly, shorten the time for the clinician to diagnose the disease condition of the patient, speed up the implementation of therapeutic measures to the patient, and have high sensitivity and good specificity.
Disclosure of Invention
In view of this, in a first aspect, the present invention provides a composition for the detection of an enteric-associated pathogen comprising:
an upstream primer, a downstream primer and a probe for detecting the shigella dysenteriae shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting vibrio parahaemolyticus as shown in SEQ ID NO. 4-6; and
an upstream primer, a downstream primer and a probe for detecting salmonella typhi are shown in SEQ ID NO. 7-9.
The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different types of pathogens by detecting different targets, so that detection and differentiation of Shigella, vibrio parahaemolyticus and salmonella typhi are simultaneously and practically produced in a single-tube reaction system, and a targeted strategy is provided for subsequent treatment. The composition has higher detection sensitivity reaching 1000 copies/mL, good specificity and more accurate detection.
Further, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.
In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is Rnase P.
In a specific embodiment, the composition further comprises an upstream primer, a downstream primer and a probe for detecting an internal standard as shown in SEQ ID NOS 10-12.
Further, the fluorophores of the probes of the compositions of the invention are different from each other and do not interfere with each other.
As used herein, "distinct and non-interfering with each other" means that the fluorophores used for each probe in the composition are different and do not affect each other's detection, i.e., can be performed using different channels. For example, ATTO 425, quasar705, FAM, HEX, ROX and CY5 can be used, which groups do not have close absorbance values and can select different channels so as not to interfere with each other.
In some specific embodiments, the fluorescent reporter group of the shigella-like probe is FAM; the fluorescent reporter group of the vibrio parahaemolyticus probe is HEX; the fluorescent reporter group of the salmonella typhi probe is ROX; the fluorescent reporter group of the internal standard is CY5.
Further, in some embodiments, the compositions of the present invention may include one or more of the above-described primer and probe pairs simultaneously. In the present invention, "pair" refers to matched upstream and downstream primers and probes that detect a target.
The compositions of the invention can be combined in any combination to detect any combination of 4 targets. Those skilled in the art can combine the primers and probe pairs as necessary to detect which targets are the corresponding targets. These combinations are included in the present invention.
For example, any 3 pairs of the 4 pairs of primers and probes may include any 2 pairs of the 4 pairs of primers and probes, and may include any 1 pair of the 4 pairs of primers and probes.
In some specific embodiments, the compositions of the invention are used in fluorescent PCR.
Further, the 3' end of the probe also has a non-fluorescent quencher.
Further, the 3' -end of the probe also has a quenching group, such as BHQ1 or BHQ2.
In a specific embodiment, the 3' end of the probe is BHQ1.
In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.
In a particular embodiment, the ingredients of the composition of the invention are present in the same package.
Further, the components of the composition of the present invention are present in a mixed form.
In a second aspect, the present invention provides the use of a composition of the invention as described above for the preparation of a kit for the detection of an enteric-associated pathogen.
In a third aspect, the present invention provides a kit for detecting an enteric-associated pathogen, the kit comprising a composition of the invention as described above.
Further, the kit also comprises a negative quality control and a positive quality control.
In a specific embodiment, the negative quality control is DEPC H 2 O, normal saline and an internal standard gene. The positive quality control substance is at least one of fragment plasmid or fragment DNA of Shigella, vibrio parahaemolyticus and Salmonella typhi.
Further, the kit also comprises dNTP, PCR buffer solution and Mg 2+ At least one of them.
Still further, the kit further comprises: at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a DNA polymerase.
Further, the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTP, dUTP, UNG enzyme, DNA polymerase, PCR buffer solution and Mg 2+ At least one of them.
Further, the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.
In a specific embodiment, the kit of the invention comprises Taq enzyme, mg 2+ dNTP (U) s, primers, probes and PCR buffer.
Common PCR buffer consists of Tris-HCl and MgCl 2 Buffer systems such as KCl and Triton X-100. The total volume in a typical single PCR reaction tube is 20. Mu.l to 200. Mu.l.
In a specific embodiment, the kit of the invention is compatible with digital PCR amplification systems, i.e., can be used directly on a digital PCR instrument for amplification.
In a fourth aspect, there is provided a method for detecting an entero-related pathogen for non-diagnostic purposes, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
In the present invention, the sample for detection may be feces, intestinal secretions, or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
UNG enzyme reaction, wherein the temperature is 50 ℃, the time is 1-5 min, and 1 cycle is performed; activating Taq enzyme at 95 ℃ for 1-5 min for 1 cycle; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
In a specific embodiment, there is provided a use for preparing a composition for detection of an enteric-associated pathogen, the detection comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
UNG enzyme reaction, wherein the temperature is 50 ℃, the time is 1-5 min, and 1 cycle is performed; activating Taq enzyme at 95 ℃ for 1-5 min for 1 cycle; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
Drawings
FIG. 1 is a graph showing the results of detection of a composition of the present invention (Shigella dysenteriae, vibrio parahaemolyticus, salmonella typhi, internal standard, respectively);
FIG. 2 is a graph of the results of the specificity of the compositions of the present invention;
FIG. 3 is a diagram showing the results of single detection of a primer probe of Vibrio parahaemolyticus according to the comparative example of the present invention;
FIG. 4 is a diagram showing the results of a four-joint detection of a primer probe of Vibrio parahaemolyticus according to the comparative example of the present invention.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Example 1, primers and probes used in the present invention
The primers and probes used in the present invention are shown in Table 1 below.
TABLE 1
Wherein, the fluorescent reporter group of the shigella-like probe is FAM; the fluorescent reporter group of the vibrio parahaemolyticus probe is HEX; the fluorescent reporter group of the salmonella typhi probe is ROX; the fluorescent reporter group of the internal standard is CY5.
Example 2 method for detecting pathogens
Fluorescent PCR amplification reaction solution: contains PCR buffer solution, taq enzyme and Mg 2+ Dntps, primers, probes, and the like. The specific reaction system is shown in Table 2. The enzyme mix consisted of 1. Mu. L H-Taq enzyme (15U/. Mu.L) mixed with 1. Mu.L RT enzyme (2. Mu.L per human).
TABLE 2
| The components | Volume/concentration in each reaction |
| PCR buffer | 19.2μL |
| dNTP(T)s | 0.5μL |
| Mg 2+ | 0.2μL |
| 0.1% DEPC Water | Is added to 40 mu L |
| Enzyme mixed solution | 2μL |
| Primer (50 pmol/. Mu.L) | 0.20μL |
| Probe (50 pmol/. Mu.L) | 0.10μL |
Reagent preparation:
and taking corresponding amounts of the PCR reaction liquid and the enzyme mixed liquid according to the quantity of the sample to be detected, the positive control and the negative control according to a proportion (38 mu L of the PCR reaction liquid/part+2 mu L of the enzyme mixed liquid/part), fully and uniformly mixing the PCR reaction liquid and the enzyme mixed liquid to form a PCR mixed liquid, and centrifuging at 2000rpm for 10s for later use.
Sample processing and sample addition
300. Mu.L of the sample to be tested, negative control, positive control were placed in a 1.5mL centrifuge tube, and nucleic acid extraction was performed using the nucleic acid extraction or purification reagent from St.Job's Biotechnology Co., ltd.
10 mu L of each of the treated sample, negative control and positive control was aspirated and added to each of the reaction tubes A-B, 40 mu L of the PCR mixture A-B was added to each of the reaction tubes, and the tube caps were closed.
The amplification reaction procedure of the SLAN-96P full-automatic medical PCR analysis system for PCR amplification according to a certain temperature and time setting procedure is shown in Table 3.
TABLE 3 Table 3
Result analysis and judgment:
if the FAM, HEX, CY and ROX channels of the A and B tubes have obvious S-shaped amplification curves and the Ct value is less than or equal to 40, judging positive; if FAM, HEX, CY and ROX channel no amplification curve (NoCt) or Ct > 40, then negative is determined as shown in Table 4:
TABLE 4 Table 4
Example 3 detection results of test samples of the inventive composition
The primers and probes shown in the example 1 are used for carrying out PCR detection on samples of Shigella dysenteriae, vibrio parahaemolyticus and salmonella typhi according to the method of the example 2, and the detection result is shown in the figure 1, so that the composition can well detect various pathogens.
Example 4 sensitivity of the composition of the invention
Using the composition of example 1 of the present invention, LOD (sensitivity) detection was performed on each target to simulate a clinical sample, and multiplex PCR detection was performed on a macrostone fluorescent quantitative PCR instrument. The results are shown in Table 5, which demonstrate that each channel can still be accurately detected for samples as low as 1000 copies/mL, indicating a sensitivity of 1000 copies/mL for the compositions of the present invention.
TABLE 5
EXAMPLE 5 specificity of the composition of the invention
In order to test the specificity of the composition in the embodiment 1 of the present invention, the specificity experiment shows that other common pathogens (bacteroides fragilis, helicobacter pylori, salmonella, staphylococcus aureus, vibrio parahaemolyticus, shigella, adenovirus, listeria monocytogenes, etc.) are tested by the method of the present invention, and the detection result is shown in fig. 3. The results show that the method of the invention has no cross-reaction to the enteric pathogens.
EXAMPLE 6 interference resistance of the composition of the invention
The test results show that a certain concentration of dexamethasone (50 mug/mL), cefmenoxime hydrochloride (50 mug/mL), zanamivir (100 mug/mL), ribavirin (100 mug/mL), azithromycin (100 mug/mL), mupirocin (50 mug/mL), tobramycin (50 mug/mL), heme (10 mug/mL), hemoglobin (10%) and other potential PCR inhibitors/interferences have no obvious influence on the test kit, and the amplification results of the composition in the presence of infectious substances are shown in the following table 7 and fig. 2.
TABLE 7
Comparative example 1, remaining poorly performing primers and probes designed according to the invention
Because of the base-pairing rules, dimers are formed between the primer and/or probe, but with little probability, this can be eliminated at the beginning of the design. However, when multiple pathogens are jointly detected, a plurality of primers and probes are arranged, dimers are easy to occur between the primers and the primers, between the probes and the probes or between the primers and the probes, so that the conservation of design (which is crucial to the accuracy of detection) is ensured, and the mutual interference among different primer probes is considered, so that the primer probes need to be carefully designed.
Thus, the inventors have also devised that the remaining primers and probes constitute a different detection system sequence, not shown, also for detecting the above-mentioned pathogens. The specific detection results are shown in fig. 3-4, and as can be seen from fig. 3, the detection effect is good when the primer probe of the comparative example vibrio parahaemolyticus is singly detected, but when the primer probe and the detection of the other 3 targets are subjected to 4 joint detection, the Ct value is obviously delayed, and the effect is poor.
Claims (10)
1. A composition for detection of an enteric-associated pathogen comprising:
an upstream primer, a downstream primer and a probe for detecting the shigella dysenteriae shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting vibrio parahaemolyticus as shown in SEQ ID NO. 4-6; and
an upstream primer, a downstream primer and a probe for detecting salmonella typhi are shown in SEQ ID NO. 7-9.
2. The composition of claim 1, wherein the first and/or second nucleic acid composition further comprises an upstream primer, a downstream primer and a probe for detecting an internal standard as shown in SEQ ID NOS 10-12.
3. The composition of claim 2, wherein the fluorophores of the probes are different from each other and do not interfere with each other.
4. The composition of claim 3, wherein the fluorescent reporter group of the shigella-like probe is FAM; the fluorescent reporter group of the vibrio parahaemolyticus probe is HEX; the fluorescent reporter group of the salmonella typhi probe is ROX; the fluorescent reporter group of the internal standard is CY5.
5. The composition according to any one of claims 1 to 4, wherein the components of the composition are present in a mixed form.
6. Use of a composition according to any one of claims 1 to 5 for the preparation of a kit for detecting an intestinal-related pathogen.
7. A kit for detecting an entero-related pathogen, the kit comprising the composition of any one of claims 1-5.
8. The kit of claim 7, further comprising a negative quality control and a positive quality control.
9. The kit of claim 7 or 8, further comprising: nucleic acid releasing reagent, nucleic acid extracting reagent, DNA polymerase, dNTP, dUTP, UNG enzyme, PCR buffer solution and Mg 2+ At least one of them.
10. Use for the preparation of a composition for the detection of an enteric-associated pathogen, said detection comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of any one of claims 1 to 5 or the kit of any one of claims 7 to 9;
3) The results were obtained and analyzed.
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