CN117802257A - Composition, kit and application for detecting pharyngitis related pathogens - Google Patents

Composition, kit and application for detecting pharyngitis related pathogens Download PDF

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CN117802257A
CN117802257A CN202311851189.0A CN202311851189A CN117802257A CN 117802257 A CN117802257 A CN 117802257A CN 202311851189 A CN202311851189 A CN 202311851189A CN 117802257 A CN117802257 A CN 117802257A
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composition
detecting
kit
probe
pharyngitis
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方红梅
程星
谭德勇
林娇阳
戴立忠
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Sansure Biotech Inc
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Abstract

The invention belongs to the field of molecular biology detection, and particularly relates to detection of related pathogens of pharyngitis, in particular to detection of group A streptococcus, EB virus, pertussis bacillus and mycoplasma pneumoniae. 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 group A streptococcus, EB virus, pertussis bacillus and mycoplasma pneumoniae 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

Composition, kit and application for detecting pharyngitis related pathogens
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to detection of pharyngitis related pathogens, and more particularly relates to group A streptococcus, EB virus, pertussis bacillus and mycoplasma pneumoniae.
Background
Group a streptococcus (Group A Streptococcus, GAS) is also known as streptococcus pyogenes, and the binding hemolysis feature is also commonly known as group a b streptococcus hemolyticus. GAS infection mainly causes acute pharyngitis, tonsillitis, scarlet fever, wind-damp heat and the like. Humans are the only host of GAS, mainly transmitted by air droplets and skin mucosal contacts. The crowd is generally susceptible to GAS, and most of the ill people are children aged 5-15 years, the old and the people with low immunity. Etiology monitoring research of 0-14 year old children acute pharyngitis in Beijing city shows that the incidence rate of 3 years of children acute GAS pharyngitis in 2012-2014 is 2685.1/10 ten thousand years. Acute rheumatic fever is an autoimmune disease caused after GAS infection, occurring 2-3 weeks after infection. The data show that the incidence rate of acute rheumatic fever in the 90 th year of the 20 th century of China is 20.05/10 ten thousand, and the incidence rate areas are greatly different.
EB virus (Epstein Barr virus, EBV), also known as human herpesvirus (Human herpes virus, HHV-4), is a double stranded DNA virus. EB virus is commonly susceptible in people, and infection can involve multiple systemic systems such as blood, respiration, urinary, digestion, nerves and the like. EB virus infection originates from oropharynx, and propagates through close contact with mouth or blood transfusion, and the main symptoms include fever, pharyngitis, cervical lymphadenectasis, etc. The EB virus infection latency period is 5-15 days, and the disease is developed all year round, and the late autumn and early winter are more. A great deal of research shows that there are a plurality of relations between EB virus and rheumatoid arthritis.
Pertussis (Bordetella pertussis, BP) is a gram-negative bacterium that causes acute respiratory illness. Pertussis is generally susceptible to the population, and the patient is the sole source of infection for the pertussis. The pertussis bacillus infection has longer disease course, symptoms such as sphagitis, fever and the like are usually displayed in the early stage, and the cough is gradually aggravated along with the development of the disease course. Patients gradually shift into a recovery period after 4 to 6 weeks, the cough is relieved, the patients tend to recover, but 1 to 10 percent of patients are easy to be infected by secondary hemolytic streptococcus and the like. The pertussis diagnosis methods which are more common clinically are a culture method and serological screening.
Mycoplasma pneumoniae (Mycoplasmal Pneumonia, MP) is a common pathogen of acute respiratory tract infection, is mainly transmitted through air droplets, and is one of the most common community-acquired pneumonia in China. Mycoplasma pneumoniae has a slow onset in a incubation period of 2 to 3 weeks, and patients have symptoms of general respiratory tract symptoms such as asymptomatic headache, pharyngalgia, fever, cough and the like, and severe cases can also cause extrapulmonary complications such as meningitis, myocarditis, pericarditis, nephritis, immune hemolytic anemia and the like. Mycoplasma pneumoniae develops all the year round, mainly in autumn and winter, and is more common in children and teenagers.
The streptococcus A, the EB virus, the pertussis bacillus and the mycoplasma pneumoniae can cause pharyngitis, and are similar in clinic, and the streptococcus A, the EB virus, the pertussis bacillus and the mycoplasma pneumoniae mainly cause symptoms such as cough, sore throat, headache, pharyngitis and the like. For the infected patient, early diagnosis and early treatment are required, and the infectious source is controlled in time to block the transmission path. A set of detection methods for rapidly diagnosing the group A streptococcus, the EB virus, the pertussis bacillus and the mycoplasma pneumoniae are established, and the detection method has important significance for differential diagnosis of the pharyngitis-related viruses and prevention and control of epidemic situations.
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 a pathogen associated with pharyngitis, comprising:
an upstream primer, a downstream primer and a probe for detecting the group A streptococcus as shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting EB virus shown in SEQ ID NO. 4-6;
an upstream primer, a downstream primer and a probe for detecting the bordetella pertussis as shown in SEQ ID NO. 7-9; and
an upstream primer, a downstream primer and a probe for detecting mycoplasma pneumoniae as shown in SEQ ID NO. 10-12.
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 streptococcus A, EB virus, pertussis bacillus and mycoplasma pneumoniae are realized simultaneously in a single-tube reaction system, 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 GAPDH.
Further, the composition also comprises an upstream primer, a downstream primer and a probe for detecting an internal standard as shown in SEQ ID NO. 13-15.
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 group a streptococcal probe is FAM; the fluorescent reporter group of the EB virus probe is HEX (or VIC); the fluorescent reporter group of the bordetella pertussis probe is ROX; the fluorescent reporter group of the mycoplasma pneumoniae 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 5 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 4 pairs of the above 5 pairs of primers and probes may be included, any 3 pairs of the above 5 pairs of primers and probes may be included, any 2 pairs of the above 5 pairs of primers and probes may be included, or any 1 pair of the above 5 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 the composition of the invention in preparing a kit for detecting pathogens related to pharyngitis, wherein the pathogens are streptococcus A, EB virus, pertussis bacillus and mycoplasma pneumoniae.
In a third aspect, the present invention provides a kit for detecting a pathogen associated with pharyngitis, said 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, physiological saline. The positive quality control material is at least one of fragment plasmids or positive strains of group A streptococcus, EB virus, pertussis bacillus and mycoplasma pneumoniae.
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), a DNA polymerase, a 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 of detecting a pathogen associated with pharyngitis 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 respiratory tract secretion or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
removing pollution of UDG, wherein the temperature is 50-60 ℃, the time is 1-6 min, and 1 cycle is performed; pre-denaturation at 95 ℃ for 1-6 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 the use of a composition for the preparation of a reagent for detecting a pathogen associated with pharyngitis, 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:
removing pollution of UDG, wherein the temperature is 50-60 ℃, the time is 1-6 min, and 1 cycle is performed; pre-denaturation at 95 ℃ for 1-6 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.
As used herein, the term "non-diagnostic purpose" refers to information not intended to obtain whether an individual is infected with the pathogen and suffering from pharyngitis. For example, the method may be used to detect the presence of the aforementioned pathogens in a test culture (e.g., respiratory secretions) in need thereof.
Drawings
FIG. 1 shows the results of detection of the composition of the present invention (group A Streptococcus, EB virus, bordetella pertussis, mycoplasma pneumoniae);
FIGS. 2 to 5 are graphs showing the sensitivity results of the compositions of the present invention (Streptococcus group A, EB virus, bordetella pertussis, and Mycoplasma pneumoniae, 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 the precision of the compositions of the present invention (Streptococcus A, EB virus, bordetella pertussis, mycoplasma pneumoniae, respectively);
FIG. 11 is a graph showing the results of two-way assay for comparative example compositions of the present invention;
FIG. 12 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 fluorescence report group of the A group streptococcus probe is FAM; the fluorescent reporter group of the EB virus probe is HEX (or VIC); the fluorescent reporter group of the bordetella pertussis probe is ROX; the fluorescence reporter group of the mycoplasma pneumoniae probe is CY5; the fluorescence reporter group of the internal standard probe is CY5.5.
Example 2 method for detecting pathogens
Sample processing and sample addition
The treatment method comprises the following steps: nucleic acid extraction was performed using a sample releasing agent (S1013E) of Sanxiang Biotechnology Co., ltd according to the procedure described in the specification.
And (3) sucking 10 mu L of each of the treated sample, the negative control and the positive control, respectively adding the 10 mu L of each of the treated sample, the negative control and the positive control into a corresponding 0.2mL PCR reaction tube, adding 40 mu L of PCR mixed solution into each tube, and covering a tube cover.
The real-time fluorescent PCR reaction system was configured as follows in table 2:
TABLE 2
The components Volume/concentration in each reaction
PCR buffer 36.17μL
UDG enzyme 0.2μL
Taq enzyme (5U/. Mu.L) 2μL
Mg 2+ (1M) 0.11μL
dNTP(0.7mM) 0.82μL
Primer(s)(150nM) 0.5μL
Probe (150 nM) 0.2μL
Sample DNA 10μL
The PCR amplification procedure was set up as follows table 3:
TABLE 3 Table 3
Analysis of results:
the target detection signal is FAM, HEX (or VIC), ROX and CY5;
setting of Baserine: baseline is typically set to 3-15 cycles, which can be specifically adjusted according to the actual situation. The adjustment principle is as follows: the region where the fluorescent signal is more stable before exponential amplification is selected, the starting point (Start) avoids the signal fluctuation in the initial stage of fluorescent collection, and the End point (End) is reduced by 1-2 cycles compared with the sample Ct of which the exponential amplification occurs at the earliest. Setting of Threshold: setting a principle that a threshold line just exceeds the highest point of a normal negative control;
and (3) quality control:
negative control: the FAM, HEX/VIC, ROX, CY and CY5.5 channels have no Ct value or Ct > 40;
positive control: the Ct of FAM, HEX/VIC, ROX, CY and CY5.5 channels is less than or equal to 33;
the requirements are met in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.
Interpretation of 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).
TABLE 4 Table 4
Example 3 detection results of test samples of the inventive composition
The primers and probes shown in example 1 were used for PCR detection of group A streptococci, EB viruses, bordetella pertussis, mycoplasma pneumoniae and internal standard in a macrostone fluorescence quantitative PCR instrument according to the method of example 2, and the detection results are shown in FIG. 1, and it can be seen from the graph that the composition of the invention 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 at concentrations of 5000, 1000, 500, 200 copies/ml, respectively, to simulate clinical specimens, and 20 multiplex PCR assays were performed on a macrostone fluorescent quantitative PCR instrument. As a result, as shown in FIGS. 2 to 5, each channel was still accurately detected for samples as low as 500 copies/mL, indicating that the sensitivity of the composition of the present invention was 500 copies/mL.
EXAMPLE 5 specificity of the composition of the invention
The composition has no cross reaction with other pathogens similar to common pathogens of genitourinary system and infection symptoms (such as hepatitis A virus, hepatitis B virus, hepatitis C virus, rubella virus, human immunodeficiency virus type 1, escherichia coli, pseudomonas aeruginosa, human parainfluenza virus type 3, cytomegalovirus, coxsackie virus type A, haemophilus influenzae, streptococcus pneumoniae, neisseria meningitidis, mycobacterium tuberculosis and the like). The results are shown in FIG. 6, which shows that the composition of the present invention has good specificity.
EXAMPLE 6 precision of the composition of the invention
The test dilutes the positive samples to a weak positive concentration level (1.0X10 4 copies/. Mu.l) was measured for in-batch precision and inter-batch precision, 10 replicates per sample. As shown in FIGS. 7-10, the detection rate of the weak positive reference sample is 100%, and the variation Coefficient (CV) of the Ct value detected in the batch and between the batches is less than 5%, which indicates that the kit has good detection precision in the batch and between the batches.
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.
Therefore, the inventors have also devised that the remaining primers and probes constitute a different detection system (sequences shown in Table 5), and are equally useful for detecting the above pathogens. The specific detection results are shown in fig. 11-12, and it can be seen from fig. 11 that the detection effect is good in the two-joint detection system for detecting group a streptococcus and mycoplasma pneumoniae, however, the detection is affected in the four-joint detection system, and the obvious delamination of the amplification curve and the significant decrease of the fluorescence increment exist, as shown in fig. 12, to further illustrate the superiority of the composition of the present invention.
TABLE 5

Claims (10)

1. A composition for detecting a pathogen associated with pharyngitis, comprising:
an upstream primer, a downstream primer and a probe for detecting the group A streptococcus as shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting EB virus shown in SEQ ID NO. 4-6;
an upstream primer, a downstream primer and a probe for detecting the bordetella pertussis as shown in SEQ ID NO. 7-9; and
an upstream primer, a downstream primer and a probe for detecting mycoplasma pneumoniae as shown in SEQ ID NO. 10-12.
2. The composition of claim 1, further comprising an upstream primer, a downstream primer and a probe for detecting an internal standard as shown in SEQ ID NOS 13 to 15.
3. 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.
4. A composition according to claim 3, wherein the fluorescent reporter group of group a streptococcus is FAM; the fluorescent reporter group of EB virus is HEX or VIC; the fluorescent reporter group of the bordetella pertussis is ROX; the fluorescent reporter group of the mycoplasma pneumoniae 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 in the preparation of a kit for detecting a pathogen associated with pharyngitis, wherein the pathogen is group a streptococcus, epstein barr virus, bordetella pertussis, mycoplasma pneumoniae.
7. A kit for detecting a pathogen associated with pharyngitis, said kit comprising the composition of any one of claims 1 to 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, UDG enzyme, PCR buffer solution and Mg 2+ At least one of them.
10. Use of a composition for the preparation of a reagent for detecting a pathogen associated with pharyngitis, 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 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.
CN202311851189.0A 2023-12-29 2023-12-29 Composition, kit and application for detecting pharyngitis related pathogens Pending CN117802257A (en)

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