CN115725795A - A composition for combined detection of pathogens causing respiratory symptoms - Google Patents
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Abstract
The invention belongs to the field of molecular biological detection, and particularly relates to a composition, a method and application of joint inspection of influenza A virus H1N1/H3N2 strains, influenza B virus BV/BY strains, novel coronavirus variant strains BA.4/BA.5 strains and HIV. The combination of the joint inspection provided BY the invention mainly utilizes a multiple fluorescence PCR analysis method to detect different pathogens BY detecting different sites on different pathogens, thereby simultaneously realizing the detection and the differentiation of influenza A virus H1N1/H3N2 strains, influenza B virus BV/BY strains, novel coronavirus variant strain BA.4/BA.5 strains and HIV in a single-tube reaction system. Allowing different pathogens to be treated differently, thus making treatment and prevention more effective. Meanwhile, the detection sensitivity of the composition is higher and reaches 200 copies/mL, and the detection is more accurate.
Description
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to a composition, a method and application of joint inspection of influenza A virus, influenza B virus, novel coronavirus and HIV, more particularly to an H1N1/H3N2 strain of influenza A virus, a BV/BY strain of influenza B virus, a BA.4/BA.5 strain of novel coronavirus variant and a composition, a method and application of joint inspection of HIV.
Background
Based on epidemiological investigation, the incubation period of the novel coronavirus is 1-14 days, the symptoms are mainly fever, hypodynamia and dry cough, and severe patients show dyspnea, septic shock, coagulation dysfunction, multiple organ failure, metabolic acidosis which is difficult to correct and the like. One of the biggest characteristics of the new coronavirus, as an RNA virus, is its easy mutation, from variant strain Alpha (Alpha B.1.1.7) to Beta (Beta B.1.351), and then to Gamma (Gamma P.1) and Delta (Delta B.1.617.2), each mutation, the new coronavirus carries with it a stronger transmission power, the World Health Organization (WHO) has expressed in the latest global new crown epidemic situation, and BA.2 before the Ormck BA.4 and BA.5 variants have been replaced becomes the main global strain, and the ratio of the two is increasing. Both increases are associated with the presence of mutations that enhance their infectivity. And some new mutations are added on BA.4/BA.5, so that the vaccine has extremely strong infectivity and immune escape capability, and the effects of a detection reagent and a vaccine can be influenced.
Influenza viruses are single-stranded negative-strand RNA viruses of the family Orthomyxoviridae, and can be classified into types A (A), B (B), and C (C) 3, depending on the nucleoprotein and M protein antigens. Influenza a (a) viruses can infect humans, pigs, horses, and birds; influenza B (B) virus only infects humans and is less pathogenic. Human influenza is mainly caused by influenza A virus and influenza B virus, the influenza A virus has the largest harm, is mainly transmitted through a respiratory tract by droplets or aerosol, and suddenly occurs and quickly spreads in people in a short time to cause epidemics of different scales; influenza a viruses are divided into many subtypes, and up to now there are 16 subtypes for HA and 9 subtypes for NA. Wherein H1N1/H3N2 primarily infects humans.
Acquired Immune Deficiency Syndrome (AIDS) is a highly harmful infectious disease caused by infection with the Human Immunodeficiency Virus (HIV), a virus that attacks the human immune system. It takes the most important CD4T lymphocyte in human immune system as main target to destroy the cell greatly and make human body lose immune function. Therefore, the human body is easy to be infected with various diseases and malignant tumors occur, and the disease death rate is high. HIV exists in blood, semen or vaginal secretion of infected person, and is transmitted via body fluid with virus, and its latent period may be as long as several years. Respiratory symptoms are manifested as long-term cough, chest pain, dyspnea, and blood-stained sputum in severe cases. AIDS is listed as a legal infectious disease in China and is listed as one of national border health monitoring infectious diseases.
One clinical manifestation may be caused by multiple pathogens, presenting difficulties for early clinical diagnosis, and the clinical treatment modalities may differ for different viruses. While multiple infections may be present in a single individual at the same time. For susceptible viruses, it is desirable to discover, treat, and control the source of infection in a timely manner, blocking the route of transmission, as early as possible. Distinguishing these viral infections provides guidance for subsequent prevention, treatment and taking targeted measures, and there is therefore a need in the art for a related product that can simultaneously detect and distinguish between influenza a, influenza b, the novel coronavirus ba.4/ba.5 and hiv.
Disclosure of Invention
In view of the above, in a first aspect, the present invention provides a composition for the combined detection of pathogens causing respiratory symptoms, comprising:
a first nucleic acid composition:
upstream and downstream primers and probes for detecting the influenza A H1N1 as shown in SEQ ID NO 1-3; and
upstream and downstream primers and probes for detecting the influenza A H3N2 as shown in SEQ ID NO. 4-6;
a second nucleic acid composition:
upstream and downstream primers and probes for detecting the second flow BY as shown in SEQ ID NO. 7-9; and
upstream and downstream primers and probes for detecting B current BV as shown in SEQ ID NO 10-12;
a third nucleic acid composition:
13-15, upstream and downstream primers and probes for detecting novel coronavirus variant strains BA.4 and BA.5; and
a fourth nucleic acid composition:
16-18, as shown in SEQ ID NO.
The composition of the joint inspection provided BY the invention mainly utilizes a multiple fluorescence PCR analysis method to detect different pathogens BY detecting different loci on different pathogens, thereby simultaneously realizing the detection and the differentiation of influenza A virus H1N1/H3N2 strains, influenza B virus BV/BY strains and novel coronavirus variant strain BA.4/BA.5 strains in a single-tube reaction system. Allowing different pathogens to be treated differently, thus making treatment and prevention more effective. Meanwhile, the detection sensitivity of the composition is higher and reaches 200 copies/mL, and the detection is more accurate.
Further, the composition includes a fifth nucleic acid composition.
The fifth nucleic acid composition is: and detecting upstream and downstream primers and probes of the internal standard.
In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is a human housekeeping gene.
In some specific embodiments, the upstream and downstream primers and probes for detecting the internal standard are the internal standard upstream primer shown in SEQ ID NO. 19, the internal standard downstream primer shown in SEQ ID NO. 20, and the internal standard probe shown in SEQ ID NO. 21.
Further, the fluorophores of the first, second, third and fourth nucleic acid composition probes of the present invention are different from each other and do not interfere with each other.
As used herein, "different from each other and non-interfering" means that the fluorophores used in each probe in the composition are different and do not interfere with each other's detection, i.e., detection can be performed using different channels. For example, FAM, HEX, ROX and CY5 can be used, which do not have close absorbance values and can select different channels and thus do not interfere with each other.
Further, in some embodiments, the compositions of the invention may include one or more of the primer and probe pairs described above. In the present invention, "pair" refers to the matched upstream and downstream primers and probes for detecting a target.
The composition of the invention can be combined into any combination form for detecting 6 corresponding targets. One skilled in the art can combine the primers and probes to detect which targets are desired, i.e., combine the primers and probes corresponding to the targets. These combinations are included in the present invention.
For example, any 5 pairs of the above 6 pairs of primers and probes may be included, any 4 pairs of the above 6 pairs of primers and probes may be included, any 3 pairs of the above 6 pairs of primers and probes may be included, any 2 pairs of the above 6 pairs of primers and probes may be included, and any 1 pair of the above 6 pairs of primers and probes may be included.
For example, only the first nucleic acid composition may be included; including only the second nucleic acid composition; only the third nucleic acid composition; including only the fourth nucleic acid composition.
For example, different pairs of different nucleic acid compositions may also be included, and for example, 1 or 2 primers and probes in a first nucleic acid composition, and/or 1 or 2 primers and probes in a second nucleic acid composition, and/or primers and probes of a third composition, and/or primers and probes of a fourth composition may be included.
In some specific embodiments, the compositions of the invention are used in fluorescence PCR.
In a specific embodiment, the fluorescent reporter of the probe of the first nucleic acid composition is FAM; the fluorescent reporter of the probe of the second nucleic acid composition is HEX; the fluorescent reporter of the probe of the third nucleic acid composition is ROX; the fluorescent reporter of the probe of the fourth nucleic acid composition is CY5.
Further, the 3' end of the probe also has a non-fluorescent quencher.
Further, the 3' -end of the probe also has a quencher group, such as BHQ1 or BHQ2.
In a specific embodiment, the 3' end of the probe is BHQ1.
Furthermore, the dosage of the primer in the composition is 0.2-0.4 mu M; the dosage of the probe in the composition is 0.1-0.2 mu M.
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 the above-described composition of the present invention in the preparation of a kit for detecting a pathogen causing respiratory symptoms, wherein the pathogen is influenza a H1N1/H3N2 strain, influenza b BV/BY strain, a novel coronavirus variant strain ba.4/ba.5 strain.
In a third aspect, the present invention provides a kit for detecting pathogens causing respiratory symptoms, said kit comprising a composition of the invention as described above.
Further, the kit also comprises a negative quality control product and a positive quality control product.
In a specific embodiment, the negative quality control is DEPC H 2 O, physiological saline and internal standard gene pseudovirus. The positive quality control product is segment plasmid, segment RNA and pseudopathy of target genes of influenza A virus H1N1/H3N2 strain, influenza B virus BV/BY strain, novel coronavirus variant strain BA.4/BA.5 strainAt least one of poison.
Further, the kit also comprises dNTPs, PCR buffer solution and Mg 2+ At least one of (1).
Still further, the kit further comprises: at least one of a nucleic acid releasing agent, a nucleic acid extracting agent, a reverse transcriptase, and a DNA polymerase.
Further, the kit further comprises a nucleic acid release reagent, a nucleic acid extraction reagent, dNTPs, reverse transcriptase, DNA polymerase, PCR buffer and Mg 2+ At least one of (1).
Further, the concentration of the reverse transcriptase is 5U/reaction-15U/reaction, for example, the reverse transcriptase can be Neoscript RT reverse transcriptase; the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.
In a particular embodiment, the kit of the invention comprises: reverse/reverse transcriptase, taq enzyme, mg 2+ 、Mn 2+ Rnasin, dNTPs, primers, probes and PCR buffer.
Common PCR buffers are Tris-HCl, mgCl 2 And buffer systems such as KCl and Triton X-100. The total volume of a single PCR reaction tube is generally 20 to 200. Mu.L.
In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, i.e., can be directly used for amplification on a digital PCR instrument.
In a fourth aspect, there is provided a method for the co-detection of pathogens causing respiratory symptoms, the method comprising the steps of:
1) Extracting or releasing 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) Results were obtained and analyzed.
In the present invention, the sample for detection may be a pharyngeal swab, an oropharyngeal swab, blood, or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
reverse transcription at 50-60 deg.c for 3-30 min for 1 circulation; pre-denaturation of cDNA at 95 deg.c for 5-60 sec for 1 circulation; denaturation at 95 deg.C for 5-20 s, annealing at 55-60 deg.C for 10-60 s, and repeating for 30-50 times to collect fluorescence.
In a specific embodiment, there is provided a method for the co-detection of pathogens causing respiratory symptoms for non-diagnostic purposes, the method comprising the steps of:
1) Extracting or releasing 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) Results were obtained and analyzed.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
reverse transcription at 50-60 deg.c for 3-30 min for 1 circulation; pre-denaturation of cDNA at 95 deg.c for 5-60 sec for 1 circulation; denaturation at 95 deg.C for 5-20 s, annealing at 55-60 deg.C for 10-60 s, and repeating for 30-50 times to collect fluorescence.
Herein, the term "non-diagnostic purpose" refers to information that is not intended to obtain information on whether an individual is infected with influenza a H1N1/H3N2 strain, influenza b BV/BY strain, novel coronavirus variant strain ba.4/ba.5 strain, and hiv and suffers from respiratory diseases such as pneumonia. For example, the method can be used to detect the presence of the pathogen in a test culture in an experiment designed for scientific research.
Drawings
FIGS. 1 to 4 are graphs showing the results of detection of the composition of the present invention (influenza A virus H1N1/H3N2 strain, influenza B virus BV/BY strain, novel coronavirus variant strain BA.4/BA.5 strain and HIV, respectively);
FIGS. 5 to 8 are graphs showing the results of sensitivity of the composition of the present invention (influenza A H1N1/H3N2 strain, influenza B BV/BY strain, novel coronavirus variant strain BA.4/BA.5 strain and HIV, respectively);
FIG. 9 is a graph showing the results of the specificity of the composition of the present invention;
FIG. 10 is a graph showing the results of testing comparative example compositions of the present invention.
Detailed Description
The present invention will be specifically explained below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.
Example 1 primers and probes used in the present invention
TABLE 1
Wherein, the fluorescence reporter group of the H1N1/H3N2 probe is FAM; the fluorescence reporter group of the BV/BY probe is HEX; the fluorescent reporter group of the new crown BA.4/BA.5 probe is ROX; the fluorescent reporter group of the HIV probe is CY5.
Example 2 method for detecting pathogens causing respiratory symptoms
The detection sample of the invention is throat swab, sputum, alveolar lavage fluid and blood. Extracting virus nucleic acid by a magnetic bead method, and performing the following operations in a sample processing chamber:
2.1 weighing a plurality of 1.5mL centrifuge tubes according to the number of samples to be measured, and adding 300 mu L of samples into each tube;
2.2 adding 500 mul of the extraction solution 1 and 50 mul of the protease K-magnetic bead mixed solution; covering the tube cover, shaking and mixing for 30s, and heating at 60 deg.C for 10min.
2.3 standing at room temperature for 1min, performing low-speed instantaneous centrifugation, placing the centrifugal tube on a magnetic separator, and slowly sucking waste liquid (paying attention to not touch magnetic beads adsorbed on the inner side of the tube wall) after 5 min;
2.4 adding 200 mul of washing solution 1 and 600 mul of washing solution 2, shaking and mixing uniformly for 30s, and placing the centrifugal tube into a magnetic separator after low-speed instantaneous centrifugation. Magnetic suction is carried out for 3min, and the liquid is completely sucked out and discarded.
2.6 placing the centrifugal tube in a centrifuge for low-speed instantaneous centrifugation, and placing the centrifugal tube in the magnetic separator again. And magnetically attracting for 3min to completely suck up the liquid at the bottom of the tube.
2.7 adding 30-100 μ L of eluent S (80 μ L of elution is recommended), shaking and mixing uniformly for 30S, eluting the magnetic beads on the wall of the centrifugal tube to the bottom of the tube, and standing for 3min at room temperature; and (3) performing low-speed instantaneous centrifugation, placing the centrifugal tube on the magnetic separator again for magnetic attraction for 3min, and transferring the eluted nucleic acid into a clean 1.5mL centrifugal tube.
2.8 aspirate 20. Mu.L of each of the treated sample, positive control and negative control into a corresponding 0.2mL PCR reaction tube, add 30. Mu.L of PCR mixture to each tube, and cover the tube.
The real-time fluorescent PCR reaction system was configured as shown in Table 2 below:
TABLE 2
The PCR amplification program was set up as in table 3 below:
TABLE 3
And (4) analyzing results:
1) The target detection signal is FAM-1, HEX (or VIC), ROX, CY5; the detection signal of the human gene internal standard is FAM-2;
2) Setting Baseline: baseline is generally set to be 3-15 cycles, and can be adjusted according to actual conditions. The adjustment principle is as follows: selecting a region with stable fluorescence signal before exponential amplification, wherein the Start (Start) avoids signal fluctuation in the initial stage of fluorescence acquisition, and the End (End) is reduced by 1-2 cycles compared with the Ct of the sample with the earliest exponential amplification. Setting Threshold: setting a rule that a threshold value line just exceeds the highest point of a normal negative control product;
results a. Samples with a typical S-type amplification curve detected for FAM channels and Ct ≤ 40 were reported to be positive for influenza a H1N1 or H3N2 strains. B. Samples with a typical sigmoid amplification curve detected for the HEX channel and a Ct ≦ 40 were reported positive for the B stream BV or BY strains. C. Samples with a typical sigmoid amplification curve detected for the ROX channel and a Ct ≦ 40 were reported as positive for the neocoronal BA.4 or BA.5 strains. D. And (3) detecting a typical S-type amplification curve for the CY5 channel, and reporting that the sample has a Ct less than or equal to 40 and is positive for the HIV.
Example 3 test results of test specimens of the composition of the invention
The primers and the probes shown in the example 1 are used for verifying the influenza A virus H1N1/H3N2 strain, the influenza B virus BV/BY strain, the novel coronavirus variant strain BA.4/BA.5 strain and the HIV pseudovirus sample according to the method of the example 2, and the result shows that the primers and the probes can be used for detecting and distinguishing the influenza A virus H1N1/H3N2 strain, the influenza B virus BV/BY strain, the novel coronavirus variant strain BA.4/BA.5 strain and the HIV, and the detection result is shown in a graph 1-4.
Example 4 sensitivity of the compositions of the invention
LOD (sensitivity) detection was performed on each target using the composition of the present invention in example 1 to simulate clinical samples for multiplex PCR detection on a fluorite fluorescent quantitative PCR instrument. The results are shown in FIGS. 5-8, which indicate that each channel can still be accurately detected for samples as low as 200 copies/mL, indicating that the sensitivity of the compositions of the present invention is 200 copies/mL.
Example 5 specificity of the compositions of the invention
To test the specificity of the composition of example 1 of the present invention, the assay was carried out using coronaviruses (NL 63, HKU1, 229E, OC43), SARS coronaviruses, MERS coronaviruses, candida glabrata, streptococcus pneumoniae, serratia marcescens, escherichia coli, staphylococcus epidermidis, acinetobacter baumannii, klebsiella pneumoniae, candida tropicalis, candida krusei, enterococcus faecalis, haemophilus influenzae, staphylococcus aureus, butyric acid bacteria, enterobacter cloacae, pseudomonas aeruginosa, legionella pneumophila, clostridium botulinum, micrococcus luteus, rhodococcus equi, listeria formates, acinetobacter johnsonii, haemophilus parainfluenza, influenza A, influenza B, and Neisseria meningitidis as samples according to the procedure described above. The results are shown in fig. 9, and all target channels have no non-specific amplification, and the specificity of the kit is good.
Comparative example 1 primers and probes designed according to the invention with the remaining Effect not good
Because of the base complementary pairing principle, a dimer is formed between the primer and (or) the probe, but the probability is very small, and the dimer can be excluded at the beginning of the design. However, when multiple pathogens are jointly detected, a large number of primers and probes are provided, dimers are easily generated between the primers and the primers, between the probes and the probes, the designed conservativeness is ensured (the conservativeness is important for the detection accuracy), and the mutual interference between different primer probes is considered, so that the primer probes need to be designed elaborately.
Therefore, the inventor also designs other primers and probes (sequences are not shown) to form different detection systems, and the other primers and probes are also used for detecting influenza A virus H1N1/H3N2 strains, influenza B virus BV/BY strains, novel coronavirus variant strains BA.4/BA.5 strains and HIV. The specific detection results are shown in FIG. 10, which shows that the overall detection effect is poor since the fluorescence value of the amplification curve is lower than the Ct value.
Claims (10)
1. A composition for the combined detection of pathogens causing respiratory symptoms comprising:
first nucleic acid composition:
1-3, as shown in SEQ ID NO, and detecting the upstream and downstream primers and the probe of the first-class H1N 1; and
upstream and downstream primers and probes for detecting the influenza A H3N2 as shown in SEQ ID NO. 4-6;
a second nucleic acid composition:
upstream and downstream primers and probes for detecting the second flow BY as shown in SEQ ID NO. 7-9; and
upstream and downstream primers and probes for detecting B current BV as shown in SEQ ID NO 10-12;
a third nucleic acid composition:
upstream and downstream primers and probes for detecting novel coronavirus variant strains BA.4 and BA.5 as shown in SEQ ID NO. 13-15; and
a fourth nucleic acid composition:
16-18, as shown in SEQ ID NO, and a probe and an upstream primer for detecting the AIDS virus.
2. The composition of claim 1, wherein the fluorophores of the first, second, third, and fourth nucleic acid composition probes are different from each other and do not interfere with each other.
3. The composition of claim 2, wherein the fluorescent reporter of the probe of the first nucleic acid composition is FAM; the fluorescent reporter of the probe of the second nucleic acid composition is HEX; the fluorescent reporter of the probe of the third nucleic acid composition is ROX; the fluorescent reporter of the probe of the fourth nucleic acid composition is CY5.
4. A composition according to any one of claims 1 to 3, characterized in that the components of the composition are present in a mixed form.
5. The composition of claim 4, further comprising a fifth nucleic acid composition comprising upstream and downstream primers and probes for detecting an internal standard.
6. The composition of claim 4, wherein the upstream and downstream primers and probe for internal standard detection are an internal standard upstream primer shown in SEQ ID NO. 19, an internal standard downstream primer shown in SEQ ID NO. 20, and an internal standard probe shown in SEQ ID NO. 21.
7. Use of a composition according to any one of claims 1 to 6 in the manufacture of a kit for the combined detection of pathogens causing respiratory symptoms.
8. A kit for the combined detection of pathogens causing respiratory symptoms, said kit comprising a composition according to any one of claims 1 to 6.
9. The kit of claim 8, further comprising a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTPs, reverse transcriptase, uracil glycosylase, DNA polymerase, PCR buffer, and Mg 2+ At least one of (a).
10. A method for the co-detection of pathogens causing respiratory symptoms for non-diagnostic purposes, said method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be detected;
2) Performing a 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 any one of claims 8 to 9;
3) Results were obtained and analyzed.
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