CN116042921A - Composition, kit and method for detecting viral encephalitis virus and application of composition - Google Patents
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Abstract
The invention belongs to the field of molecular biology detection, and particularly relates to detection of mumps virus, human parvovirus B19 and human double-Epstein-Barr virus. The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different viruses by detecting targets on different viruses, so that detection and distinction of mumps virus, human parvovirus B19 and human double-Epstein-Barr virus are realized in a single-tube reaction system. Therefore, the method realizes clear pathogen, early diagnosis and false positive prevention, and enables different viruses to be treated in a targeted manner. Meanwhile, the composition provided by the invention has higher detection sensitivity, achieves 600 copies/mL and is more accurate in detection.
Description
Technical Field
The invention belongs to the field of molecular biology detection, and particularly relates to detection of mumps virus, human parvovirus B19 and human double-Epstein-Barr virus.
Background
Mumps virus (MuV) belongs to paramyxoviridae in taxonomic, is single-strand negative-strand RNA, has only one serotype, is transmitted through droplets in people, is a causative agent of Mumps, which is a common acute respiratory infectious disease in children, and is mainly prevalent in winter and spring for two seasons. Mumps can involve other organs in addition to salivary glands, producing other complications, with a 1% to 10% chance of meningitis occurring in the patient, typically manifested as headache, vomiting, cervical rigidity, and cerebrospinal lymphocytosis, even seizures or coma.
Human parvovirus B19 (Human parvovirus B, B19) is a linear single-stranded non-enveloped small DNA virus with the smallest volume and the simplest structure in animal viruses, belongs to members of the genus rhodoviras of the family parvoviridae in taxonomy, can cause various childhood diseases through respiratory tract infection, has higher infection rate and morbidity rate, and is also a main transmitter. The research shows that the human parvovirus B19 can cause asymptomatic infection and symptomatic infection, has wide and complex clinical manifestation spectrum including infectious erythema, blood system diseases, related arthritis and the like, and simultaneously the disease spectrum caused by the human parvovirus B19 is continuously expanded, including bronchitis, encephalitis and the like, and a certain hospital successfully diagnoses an encephalitis patient caused by the human parvovirus B19 in 2020.
Human double-Epstein-Barr virus (Human parechovirus, HPeVs) was a single-stranded positive-strand RNA virus of the genus double-Epstein-Barr in the family Picornaviridae, and was first isolated from the stool of diarrhea patients by Japanese scientists in 1956, 14 human double-Epstein-Barr genotypes have been found to date. Human double-Epstein-Barr virus is easy to infect and epidemic in young children, and the generated symptoms are similar to human enteroviruses, mainly cause mild symptoms such as gastrointestinal tract, respiratory tract and the like, but for example, encephalitis, myocarditis and central nervous system symptoms are also reported; in one study of 581 human double-Epstein-Barr virus infected individuals by WHO, 12% of the patients were found to develop CNS symptoms.
Viral encephalitis (Virus Encephalitis) is an intracranial acute inflammatory disease caused by various viruses, which can affect the meninges and brain parenchyma, and causes various pathogen types of viral encephalitis, and is one of the main diseases seriously affecting the world public health. Mumps virus, human parvovirus B19 and human bieicovirus are all causative agents of viral encephalitis, and the clinical manifestations, the disease states and the prognosis of viral encephalitis caused by different viruses are different. Because the pathogen causing severe viral encephalitis has the characteristics of strong infection, wide transmission range, high morbidity and mortality, etc., measures need to be taken as early as possible to confirm the type of the pathogen causing encephalitis, and corresponding treatment measures and prognosis measures are taken for specific pathogens, so that viral encephalitis is fundamentally prevented.
Therefore, there is a strong need in the art for a simple, rapid and objective product for detecting the above viruses, so as to achieve clear pathogen, early diagnosis and avoid false positive results, and enable different viruses to be treated in a targeted manner.
Disclosure of Invention
In view of this, in a first aspect, the present invention provides a composition for detecting viral encephalitis virus, comprising:
upstream and downstream primers and probes for detecting mumps virus as shown in SEQ ID NO. 1-3;
an upstream primer and a downstream primer for detecting the human parvovirus B19 and a probe shown in SEQ ID NO. 4-6; and
the upstream and downstream primers and probes for detecting the human double-Epstein-Barr virus are shown as SEQ ID NOs 7-9.
The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different viruses by detecting targets on different viruses, so that detection and distinction of mumps virus, human parvovirus B19 and human double-Epstein-Barr virus are realized in a single-tube reaction system. Therefore, the method realizes clear pathogen, early diagnosis and false positive prevention, and enables different viruses to be treated in a targeted manner. Meanwhile, the composition provided by the invention has higher detection sensitivity, achieves 600 copies/mL and is more accurate in detection.
Further, the composition includes an upstream and 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 a human housekeeping gene.
In some specific embodiments, the composition further comprises upstream and downstream primers and probes 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 mumps virus probe is FAM; the fluorescent reporter group of the human parvovirus B19 probe is ROX; the fluorescent reporter group of the human double-Epstein-Barr virus probe is CY5; the fluorescent reporter group of the internal standard probe is HEX (or VIC).
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.
Further, the amount of the primer in the composition is 0.05 to 5. Mu.M; the amount of probe in the composition is 0.05 to 5. 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 invention provides the use of the composition of the invention described above in the preparation of a kit for combined detection and differentiation of viral encephalitis viruses, wherein the viruses are mumps virus, human parvovirus B19 and human Bieovirus.
In a third aspect, the invention provides a kit for combined detection and differentiation of viral encephalitis viruses, the kit comprising the 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 internal standard gene pseudovirus. The positive quality control is at least one of mumps virus, human parvovirus B19 and human double-Epstein-Barr virus fragment plasmid, fragment RNA or fragment DNA, and 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, a reverse transcriptase and a DNA polymerase.
Further, the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTPs, a reverse transcriptase, a DNA polymerase, RNasin, a PCR buffer solution and Mg 2+ At least one of them.
Further, the concentration of the reverse transcriptase is 5U/reaction-15U/reaction, for example, the reverse transcriptase may be Neoscript RT reverse transcriptase or MMLV enzyme; the concentration of the DNA polymerase is 3U/reaction-15U/reaction, for example, the DNA polymerase may be Taq enzyme.
In a specific embodiment, the kit of the invention comprises: reverse transcriptase, taq enzyme, mg 2+ Rnasin, dNTPs, 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 joint inspection and differentiation of viral encephalitis viruses, 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 serum, blood, or the like, but is not limited thereto.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
reverse transcription is carried out at 50-60 ℃ for 3-30 minutes for 1 cycle; activating Taq enzyme at 95 deg.c for 5-120 sec for 1 circulation; 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, a method for detecting and differentiating viral encephalitis viruses for non-diagnostic purposes is provided, 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:
reverse transcription is carried out at 50-60 ℃ for 3-30 minutes for 1 cycle; activating Taq enzyme at 95 deg.c for 5-120 sec for 1 circulation; 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 virus described above and suffering from viral encephalitis, and the like. For example, the method can be used for detecting the presence or absence of the virus in a detection culture (e.g., tissue fluid, spinal fluid, etc.) in experiments for scientific research.
Drawings
FIG. 1 is a graph of the results of testing compositions of the present invention;
FIGS. 2-4 are graphs showing the sensitivity test results of the compositions of the present invention (mumps virus, human parvovirus B19 and human Bieovirus, respectively);
FIG. 5 is a graph showing the results of the detection of the composition of 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
TABLE 1
Wherein, the fluorescence report group of the MuV probe is FAM; the fluorescent reporter group of the B19 probe is ROX; the fluorescent reporter group of HPeVs is CY5; the fluorescent reporter group for GAPDH is HEX (VIC).
Example 2 method for detecting viral encephalitis Virus
Fluorescent PCR amplification reaction solution: contains PCR buffer solution and Mg 2+ dNTP, rnasin, TE buffers, primers, probes, etc. The reaction system in this example is shown in Table 2:
TABLE 2
Preparing an enzyme mixed solution:
the enzyme mixture consists of Neoscript RT reverse transcriptase and H-Taq enzyme. H-Taq enzyme (5U/. Mu.L) and Neoscript RT enzyme (5U/. Mu.L) were mixed in a certain ratio (3. Mu. L H-Taq enzyme per human being was mixed with 1. Mu.L Neoscript RT enzyme).
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 (36 mu L of the PCR reaction liquid/4 mu L of the enzyme mixed liquid/human 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 adding:
200 mu L of a sample to be detected, negative control and positive control are 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 operation of the instruction book; 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.
And (3) PCR amplification:
and (3) performing PCR amplification according to a certain temperature and time setting program on the SLAN-96P full-automatic medical PCR analysis system. The preferred embodiment of the present invention is shown in Table 3.
TABLE 3 Table 3
Interpretation of test results:
if the sample FAM, HEX (VIC), ROX and CY5 channels have obvious S-shaped amplification curves and Ct value is less than or equal to 40, judging positive; if the sample FAM, ROX and CY5 channels have No amplification curve (No Ct) or Ct value >40, and HEX (VIC) internal standard channels are positive (Ct value is less than or equal to 40), the sample FAM, ROX and CY5 channels are judged to be negative. The details are shown in Table 4 below.
TABLE 4 Table 4
Example 3 detection results of test samples of the inventive composition
The primer and the probe shown in the example 1 are verified according to the method of the example 2, and the results show that the composition can carry out joint inspection and differentiation on mumps virus/human parvovirus B19/human double-angstrom virus and the detection result is shown in figure 1.
Example 4 sensitivity of the composition of the invention
LOD (sensitivity) detection was performed on each target using the composition of example 1 of the present invention to simulate clinical samples, and multiplex PCR detection was performed on SLAN-96P full-automatic medical PCR analysis system. The 100% detection limit of the kit on mumps virus, human parvovirus B19 and human double Epstein-Barr virus is 600.0copies/mL (see Table 5, figures 2-4).
TABLE 5
EXAMPLE 5 specificity of the composition of the invention
Experiments were performed on common viral encephalitis pathogens (Coxsackie virus, poliovirus, chikungunya virus, herpes simplex virus, human double Epstein-Barr virus, varicella-zoster virus, cytomegalovirus, forest encephalitis virus, japanese encephalitis virus, human parvovirus B19, measles virus, etc.). The results show that the method of the invention has no cross reaction to the viral encephalitis pathogen. See in particular table 6 below.
TABLE 6
EXAMPLE 6 interference resistance and stability of the inventive composition
The test results show that potential PCR inhibitors/interfering substances such as dexamethasone (50 mug/mL), cefmenoxime hydrochloride (50 mug/mL), zanamivir (100 mug/mL), ribavirin (100 mug/mL), azithromycin (100 mug/mL), histamine hydrochloride (200 mug/mL), beclomethasone (50 mug/mL), mupirocin (50 mug/mL), tobramycin (50 mug/mL), mometasone (50 mug/mL), fluticasone (50 mug/mL), budesonide (50 mug/mL), triamcinolone acetonide (100 mug/mL), heme (10 mug/mL), purified mucin (20 mug/mL), absolute ethyl alcohol (20%V/V) and the like have no obvious influence on the kit. See in particular table 7 below.
TABLE 7
The kit is detected after being stored for 11 months under the actual storage condition (-20+/-5 ℃), and has stable performance; the test result of the accelerated stability of the damage at 37 ℃ shows that the kit is stored in a constant temperature box at 37 ℃ for 24 hours, and the result meets the quality requirement; the freeze thawing stability test shows that the different kit passes through freeze thawing once at each detection time point at the actual storage temperature, and is continuously detected for 4 times, and the results all meet the quality requirements.
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 viruses are detected in a combined way, 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 designed carefully.
Therefore, the inventors have designed other primers and probes (sequences not shown) to form different detection systems, and the detection systems are also used for detecting the viruses, and specific detection results are shown in fig. 5, which shows that the detection systems only have partial amplification curves, the amplification efficiency is obviously reduced, and other targets exist without amplification curves, so that the overall detection effect is poor.
Claims (10)
1. A composition for detecting a viral encephalitis virus comprising:
upstream and downstream primers and probes for detecting mumps virus as shown in SEQ ID NO. 1-3;
an upstream primer and a downstream primer for detecting the human parvovirus B19 and a probe shown in SEQ ID NO. 4-6; and
the upstream and downstream primers and probes for detecting the human double-Epstein-Barr virus are shown as SEQ ID NOs 7-9.
2. The composition of claim 1, further comprising upstream and downstream primers and probes for detecting an internal standard.
3. The composition of claim 2, further comprising upstream and downstream primers and probes for detecting an internal standard as shown in SEQ ID NOS.10-12.
4. The composition of claim 1, wherein the fluorescent reporter group of the mumps virus probe is FAM; the fluorescent reporter group of the human parvovirus B19 probe is ROX; the fluorescent reporter group of the human double-Epstein-Barr virus probe is CY5; the fluorescent reporter group of the internal standard probe is HEX.
5. The composition of claim 1, 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 combined detection and differentiation of viral encephalitis viruses, wherein the viruses are mumps virus, human parvovirus B19 and human bieicovirus.
7. A kit for joint detection and differentiation of viral encephalitis viruses, comprising the composition of any 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, reverse transcriptase, DNA polymerase, RNasin, dNTP, PCR buffer and Mg 2+ At least one of them.
10. A method for detecting and differentiating viral encephalitis viruses 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 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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