CN117802253A - Composition, kit and application for detecting infection-related pathogens - Google Patents
Composition, kit and application for detecting infection-related pathogens Download PDFInfo
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Abstract
The invention belongs to the field of molecular biological detection, and particularly relates to detection of infection related pathogens, and more particularly relates to detection of benakokra, influenza virus, typhoid bacillus and brucella. 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 benakoox, influenza virus, typhoid bacillus and brucella are realized in a single-tube reaction system. The composition has higher detection sensitivity reaching 500 copies/mL, good specificity and more accurate detection.
Description
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to detection of infection-related pathogens, and more particularly relates to a bernakex body, influenza virus, typhoid bacillus and brucella.
Background
Q Fever (Q Fever) is an acute natural epidemic infectious disease caused by Coxiella burn, the incubation period is generally about 3 weeks, and the clinical manifestations of the disease are various, including acute, chronic, subclinical manifestations and the like, and are mainly manifested by symptoms such as aversion to cold, fever, severe headache, muscle pain and the like. The clinical manifestation of chronic Q heat is atypical, the conventional detection has low positive rate, the curative effect of the empirical antibacterial medicament is poor, the chronic Q heat is easy to ignore, and the misdiagnosis rate and the missed diagnosis rate are high.
Influenza (Influenza) is a common acute respiratory infectious disease caused by Influenza virus (Influenza virus), and is extremely high in infectivity and transmission speed in winter and spring. Influenza viruses can be classified into four types, a, b, c, and t, with influenza a and b being more common. Symptoms of influenza include fever, cough, runny nose, sore throat, headache, muscle pain, hypodynamia and the like, and severe cases can have complications such as pneumonia, myocarditis, encephalitis and the like. The incubation period of influenza is generally 1-3 days, so that the influenza has strong infectivity and high transmission speed, and can cause large-scale epidemic in a short time.
Typhoid (Typhoid river) is an acute intestinal infectious disease caused by Typhoid bacillus (Salmonella enterica), and is mainly transmitted through diet, drinking water and other ways. Latency is typically 7-14 days, symptoms include fever, headache, nausea, vomiting, abdominal pain, diarrhea, etc., and complications such as intestinal bleeding, intestinal perforation, etc. may occur in severe cases.
Brucellosis (Brucellosis) is transmitted by Brucella (Brucella) through damaged skin mucosa, digestive tract, respiratory tract and other pathways. Invade the body, and cause infectious diseases which are common to people and livestock with infectious-allergic reaction. Acute cases are mainly manifested by fever, weakness, hyperhidrosis, muscle, arthralgia, and hepatosplenomegaly and lymphadenopathy. Chronic cases often manifest as joint damage and the like, and can severely lose work capacity. If the treatment is not in time, the treatment is easy to be changed into chronic treatment, and the chronic treatment is difficult to cure.
Q fever, influenza, typhoid fever and brucellosis can cause symptoms such as fever, headache, hypodynamia, muscle soreness and the like, are similar clinically, and can cause serious complications for serious patients. For the infected patient, early diagnosis and accurate diagnosis are needed, symptomatic treatment is achieved, and complications caused by misdiagnosis and missed diagnosis are avoided as much as possible. A set of detection methods for rapidly diagnosing Q heat, influenza (containing A and B), typhoid and brucellosis are established, and the detection method has important significance for differential diagnosis of related diseases and prevention and control of epidemic situation.
Therefore, there is a need in the art for a product that can simply and rapidly detect the above pathogens, has high sensitivity and good specificity, provides a relatively sufficient basis for rapid diagnosis and elimination of infection by different pathogens for a clinician, shortens the time for diagnosis of a patient's condition by a clinician, and accelerates the implementation of therapeutic measures to a patient.
Disclosure of Invention
In view of this, in a first aspect, the present invention provides a composition for detecting an infection-associated pathogen comprising:
an upstream primer, a downstream primer and a probe for detecting the benacox body as shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting influenza virus as shown in SEQ ID NO. 4-9;
an upstream primer, a downstream primer and a probe for detecting typhoid bacillus shown in SEQ ID NO. 10-12; and
the upstream primer, the downstream primer and the probe for detecting the Brucella are shown as SEQ ID NO. 13-15.
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 benacox, influenza virus, typhoid bacillus and brucella are realized in a single-tube reaction system at the same time, and a targeted strategy is provided for subsequent treatment. The composition has higher detection sensitivity reaching 500 copies/mL, good specificity and more accurate detection, provides a basis for a clinician to diagnose and eliminate different pathogen infections more fully and rapidly, shortens the time of the clinician to diagnose the illness state of a patient, and quickens the implementation of treatment measures to the patient.
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.
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 benakoox probe is FAM; the fluorescent reporter group of the influenza virus probe is HEX (or VIC); the fluorescence reporter group of the typhoid bacillus probe is ROX; the fluorescent reporter group of the Brucella probe 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 above 4 pairs of primers and probes may be included, any 2 pairs of the above 4 pairs of primers and probes may be included, and any 1 pair of the above 4 pairs of primers and probes may be included.
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 invention provides the use of a composition of the invention as described above in the manufacture of a kit for detecting a pathogen associated with infection, wherein the pathogen is benakokra, influenza virus, typhoid bacillus, brucella.
In a third aspect, the present invention provides a kit for detecting an infection-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 controlThe product is DEPC H 2 O, physiological saline. The positive quality control agent is at least one of Benacox, influenza virus, typhoid bacillus, brucella fragment plasmid or positive strain (pseudovirus).
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, dNTPs, dUTP, uracil glycosylase (UDG), DNA polymerase, reverse transcriptase, PCR buffer, and Mg 2+ At least one of them.
Further, the concentration of the DNA polymerase is 5U/reaction to 20U/reaction, for example, the DNA polymerase may be Taq enzyme.
In a specific embodiment, the kit of the invention comprises Taq enzyme, mg 2+ dNTPs (T), 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 30. Mu.l to 50. Mu.l.
In a fourth aspect, there is provided a method of detecting an infection-associated pathogen for non-diagnostic purposes, the method comprising the steps of:
1) Extracting nucleic acid of a sample to be detected;
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 a pharyngeal swab, sputum, blood, or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
reverse transcription, the temperature is 50-60 ℃, the time is 3-20 min, and 1 cycle is performed; pre-denaturation at 90-95 deg.c for 30-60 sec for 1 circulation; denaturation at 90-95 deg.c for 1-20 sec, annealing at 55-60 deg.c for 10-30 sec, 40-45 cycles, and collecting fluorescence.
In a specific embodiment, there is provided the use of a composition for the preparation of a reagent for detecting an infection-associated pathogen, said detection comprising the steps of:
1) Extracting nucleic acid of a sample to be detected;
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:
reverse transcription, the temperature is 50-60 ℃, the time is 3-20 min, and 1 cycle is performed; pre-denaturation at 90-95 deg.c for 30-60 sec for 1 circulation; denaturation at 90-95 deg.c for 1-20 sec, annealing at 55-60 deg.c for 10-30 sec, 40-45 cycles, and collecting fluorescence.
In this context, the term "non-diagnostic purpose" refers to information not intended to obtain whether an individual is infected with the pathogen and suffering from the infection. For example, the method may detect the presence of the aforementioned pathogens in culture (e.g., blood, interstitial fluid, respiratory secretions, etc.) as desired.
Drawings
FIG. 1 shows the results of the detection of the compositions of the invention (Benacox, influenza, typhoid, brucella);
FIGS. 2 to 5 are graphs showing the sensitivity results of the compositions of the present invention (Benacox, influenza, typhoid, brucella, respectively);
FIG. 6 is a graph of the results of the specificity of the compositions of the present invention;
FIGS. 7 to 10 are graphs showing the results of single detection of the comparative example composition of the present invention (Benacox, influenza, typhoid, brucella, respectively);
FIG. 11 is a graph showing the results of a four-joint test of the comparative example composition 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 benakokra probe is FAM; the fluorescent reporter group of the influenza virus probe is HEX (VIC); the fluorescence reporter group of the typhoid bacillus is ROX; the fluorescent reporter group of brucella is CY5.
Example 2 method for detecting pathogens
Sample processing and sample adding:
300. Mu.L of the simulated sample (pseudovirus, plasmid), negative control and positive control to be tested are respectively taken into a 1.5mL centrifuge tube, and nucleic acid extraction is carried out by using a nucleic acid extraction or purification reagent of Sanxiang biotechnology Co., ltd according to the specification operation of the reagent for later use.
Referring to Table 2, 20. Mu.L of each of the above-mentioned processed analog sample nucleic acid to be tested, negative control and positive control, and other components were sucked up, respectively, and added into corresponding 0.2mL PCR reaction tubes, and mixed well to obtain a PCR mixture, and covered with a tube cap.
TABLE 2
| The components | Each of which is invertedVolume/concentration in the stress |
| PCR buffer | 23μL |
| RT enzyme (5U/. Mu.L) | 0.3μL |
| Taq enzyme (5U/. Mu.L) | 3μL |
| Mg2+(1M) | 0.2μL |
| dNTPs(T)(0.7mM) | 2μL |
| Primer (150 nM) | 1μL |
| Probe (150 nM) | 0.5μL |
| Sample nucleic acid | 20μL |
And (3) PCR amplification:
PCR amplification is performed by a PCR instrument such as the biological system SLAN96 according to a predetermined temperature and time setting program. The preferred embodiment of the present invention is shown in Table 3.
TABLE 3 Table 3
Analysis of results:
and after the reaction is finished, automatically storing the result, and respectively analyzing the amplification curves of the detection targets. And (3) adjusting the Start value, end value and Threshold value of Baseline according to the analyzed image (a user can adjust the Start value at 3-15, the End value can be set at 5-20, the amplification curve of the negative control is adjusted to be straight or lower than a Threshold line), clicking Analyze for analysis, so that various parameters meet the requirements in quality control, and recording qualitative results under a Plate window.
Quality control
Negative control: the FAM, ROX, HEX/VIC and CY5 channels have no Ct value;
positive control: the Ct of the FAM, ROX, HEX/VIC and CY5 channels is less than or equal to 35;
the requirements are met in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.
Positive judgment value
The Ct reference value of the target gene detected by the kit is determined to be 40 through the research of the reference value.
Interpretation of test results
Evaluation of the test results of the clinical specimens should be performed after the positive and negative controls are tested and determined to be valid and acceptable. If the quality control is invalid, the result of the patient cannot be explained. The following table describes the interpretation of the results regarding the use of the above quality control. The end user needs to check the fluorescence curve before final interpretation. For weak positive samples, all curves are typical S-shaped amplification curves or plateau-free phases (38.ltoreq.Ct.ltoreq.40).
Interpretation of results
TABLE 4 Table 4
Note that Ct value >40 or not detected as negative (-); ct value is less than or equal to 40 and positive (+).
Example 3 detection results of test samples of the inventive composition
The primers and probes shown in example 1 were used to perform PCR detection on a mixed analog sample of Benacox, influenza virus, typhoid bacillus and Brucella in the method of example 2 on a macrostone fluorescence quantitative PCR instrument, and the detection results are shown in FIG. 1, and it can be seen from the graph that the composition of the invention can perform good detection on 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 at concentrations of 1000, 800, 500, 200 copies/ml, respectively, to simulate clinical specimens, and 20 multiplex PCR assays were performed on a macrostone fluorescent quantitative PCR instrument. The results are shown in FIGS. 2-5, which show that each channel can still be accurately detected for samples as low as 500 copies/mL, and the detection rate is 100%, which shows that the sensitivity of the composition of the invention is 500 copies/mL.
EXAMPLE 5 specificity of the composition of the invention
The composition of the invention has no cross reaction with common pathogens (measles virus, mumps virus, rubella virus, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, type I human parainfluenza virus, cytomegalovirus, enterovirus, human interstitial pneumovirus, pertussis bacillus, chlamydia pneumoniae, haemophilus influenzae and haemophilus influenzae). The results are shown in FIG. 6, which shows that the composition of the present invention has good specificity.
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 devised that the remaining primers and probes constitute a different detection system (sequences not shown) for detecting the above pathogens as well. Specific detection results are shown in fig. 7-10, and it can be seen from the figures that the detection effect of the primers and probes for detecting 4 targets is good in a single detection system, however, the detection is affected in a four-joint detection system, and obvious layering of an amplification curve and remarkable reduction of fluorescence increment exist, as shown in fig. 11, so that the superiority of the composition of the invention is further illustrated.
Claims (10)
1. A composition for detecting an infection-associated pathogen comprising:
an upstream primer, a downstream primer and a probe for detecting the benacox body as shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting influenza virus as shown in SEQ ID NO. 4-9;
an upstream primer, a downstream primer and a probe for detecting typhoid bacillus shown in SEQ ID NO. 10-12; and
the upstream primer, the downstream primer and the probe for detecting the Brucella are shown as SEQ ID NO. 13-15.
2. The composition of claim 1, wherein the fluorophores of the probes of the composition are different from each other and do not interfere with each other.
3. The composition of claim 1, further comprising an upstream primer, a downstream primer, and a probe for detecting an internal standard.
4. A composition according to claim 3, wherein the fluorescent reporter group of benacox is FAM; the fluorescent reporter group of the influenza virus is HEX or VIC; the fluorescence reporter group of the typhoid bacillus is ROX; the fluorescent reporter group of the Brucella probe 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 infection-associated pathogen, wherein the pathogen is benakokra, influenza virus, typhoid bacillus, brucella.
7. A kit for detecting an infection-associated 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, reverse transcriptase, dNTP, dUTP, UDG enzyme, PCR buffer and Mg 2+ At least one of them.
10. Use of a composition for the preparation of a reagent for detecting an infection-associated pathogen, the detection comprising the steps of:
1) Extracting nucleic acid of a sample to be detected;
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 10 to 12;
3) The results were obtained and analyzed.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311774241.7A CN117802253A (en) | 2023-12-21 | 2023-12-21 | Composition, kit and application for detecting infection-related pathogens |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311774241.7A CN117802253A (en) | 2023-12-21 | 2023-12-21 | Composition, kit and application for detecting infection-related pathogens |
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| CN117802253A true CN117802253A (en) | 2024-04-02 |
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| CN202311774241.7A Pending CN117802253A (en) | 2023-12-21 | 2023-12-21 | Composition, kit and application for detecting infection-related pathogens |
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| Country | Link |
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| CN (1) | CN117802253A (en) |
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