CN118086584A - Composition and kit for detecting neonatal enterovirus and application of composition and kit - Google Patents
Composition and kit for detecting neonatal enterovirus and application of composition and kit Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology detection, and particularly relates to detection of neonatal enterovirus, in particular to detection of enterovirus D68, paraenterovirus A3 and Epstein-Barr virus 30. 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 the detection and the differentiation of enterovirus D68, paraenterovirus A3 and Epstein-Barr virus 30 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 biology detection, and particularly relates to detection of different types of neonatal enteroviruses, in particular to detection of enterovirus D68, paraenterovirus A3 and Epstein-Barr virus 30.
Background
Neonatal enterovirus type 68 (EV-D68) belongs to one serotype of neonatal enterovirus group D, which does not colonize the gut but infects the respiratory tract, and thus can only be isolated from the respiratory secretions of infected persons. Respiratory diseases caused by neonatal enterovirus D68 mainly occur in children, and symptoms usually resemble common cold (such as runny nose, cough, general discomfort, and feverish few occurrences). Some children, particularly those suffering from asthma, may develop severe symptoms that involve the lower respiratory tract. Infection of neonates with neonatal enterovirus D68 may manifest symptoms like sepsis such as high fever, weakness, DIC, bleeding, and multiple organ failure. And meanwhile, the damage of the central nervous system, the liver, the pancreas and the adrenal glands can be seen, and serious threat to the health of newborns exists.
Human Paracolone virus (human parechovirus, HPeV) belongs to Paracolone genus of Paracoviridae, and is a non-enveloped, single-stranded RNA virus. HpeV (HPeV 1 to 16) of 16 different subtypes have been found. Newborns are susceptible to HPeV infections, accounting for 60% -73%, of which HPeV is a common type of infection for children HPeV. HPeV3 infection is transmitted mainly by the faecal-oral and spray routes in closely contacted people, such as cross-infection between human sources, community infection, and neonatal ward HPeV fulminant infection. Children HPeV are infected with various clinical manifestations, and can be asymptomatic or manifest as mild respiratory and digestive system infection symptoms, and also can manifest as severe infections such as myocarditis, pneumonia, meningoepithymen encephalitis, and combined hemophilus syndrome.
Human Epstein-Barr virus type 30 (Echov 30) belongs to the genus Picornaviridae, is a serotype of human enterovirus in neonatal enterovirus group B, echoV has no envelope virus and can survive stably under severe environmental conditions (e.g., high salt, high pH and 37 ℃). Since humans can still expel viruses from the respiratory tract and intestinal tract within weeks after infection with human neonatal enteroviruses, the viruses can cause serious hazards such as viral transmission, infection, and even outbreak. Of the cases of neonatal viral encephalitis, the case caused by EchoV virus accounts for 80% -93%, while EchoV is the most commonly isolated neonatal enterovirus of the cases of viral encephalitis.
Therefore, there is a need in the art for a product that can simply and quickly detect the different pathogens described above, so as to provide a basis for a clinician to diagnose and eliminate infection by the different pathogens more fully and quickly, shorten the time for the clinician to diagnose the disease condition of the patient, speed up the implementation of therapeutic measures to the patient, and have high sensitivity and good specificity.
Disclosure of Invention
In view of this, in a first aspect, the present invention provides a composition for neonatal enterovirus detection comprising:
an upstream primer, a downstream primer and a probe for detecting enterovirus D68 as shown in SEQ ID NO. 1-3;
An upstream primer, a downstream primer and a probe for detecting the Paracolone virus A3 type as shown in SEQ ID NO. 4-6; and
The upstream primer, the downstream primer and the probe for detecting the type 30 of the Epstein-Barr virus are shown in SEQ ID NOs 7 to 9.
The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different types of pathogens by detecting different targets, so that the detection and the differentiation of enterovirus D68, paraenterovirus A3 and Epstein-Barr virus 30 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.
Further, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.
In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is Rnase P.
In a specific embodiment, the composition further comprises an upstream primer, a downstream primer and a probe for detecting an internal standard as shown in SEQ ID NOS 10-12.
Further, the fluorophores of the probes of the compositions of the invention are different from each other and do not interfere with each other.
As used herein, "distinct and non-interfering with each other" means that the fluorophores used for each probe in the composition are different and do not affect each other's detection, i.e., can be performed using different channels. For example, ATTO 425, quasar705, FAM, HEX, ROX and CY5 can be used, which groups do not have close absorbance values and can select different channels so as not to interfere with each other.
In some specific embodiments, the fluorescent reporter group of the enterovirus D68-type probe is FAM; the fluorescent reporter group of the paraminovirus A3 probe is HEX; the fluorescent reporter group of the Epstein-Barr virus 30 type probe is CY5; the fluorescence reporter group of the internal standard 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 4 pairs of primers and probes may include any 2 pairs of the 4 pairs of primers and probes, and may include any 1 pair of the 4 pairs of primers and probes.
In some specific embodiments, the compositions of the invention are used in fluorescent PCR.
Further, the 3' end of the probe also has a non-fluorescent quencher.
Further, the 3' -end of the probe also has a quenching group, such as BHQ1 or BHQ2.
In a specific embodiment, the 3' end of the probe is BHQ1.
In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.
In a particular embodiment, the ingredients of the composition of the invention are present in the same package.
Further, the components of the composition of the present invention are present in a mixed form.
In a second aspect, the invention provides the use of the composition of the invention in the preparation of a kit for neonatal enterovirus detection.
In a third aspect, the invention provides a kit for detecting neonatal enterovirus, the kit comprising a composition of the invention as described above.
Further, the kit also comprises a negative quality control and a positive quality control.
In a specific embodiment, the negative quality control is at least one of DEPC H 2 O, physiological saline, and an internal standard gene. The positive quality control product is at least one of enterovirus D68, paraenterovirus A3, and fragment nucleic acid of Epstein-Barr virus 30, pseudovirus, etc.
Further, the kit further comprises at least one of dNTPs, PCR buffer and Mg 2+.
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.
Still further, the kit further comprises at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTP, dUTP, UNG enzyme, reverse transcriptase, DNA polymerase, PCR buffer, and Mg 2+.
Further, the concentration of the reverse transcriptase is 5U/reaction to 15U/reaction, for example, the reverse transcriptase may be Neoscript RT reverse transcriptase; 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 reverse/reverse transcriptase, taq enzyme, mg 2+, dNTP (U) s, primers, probes and PCR buffer.
Common PCR buffers consist of Tris-HCl, mgCl 2, KCl, triton X-100 and other buffer systems. The total volume in a typical single PCR reaction tube is 20. Mu.l to 200. Mu.l.
In a specific embodiment, the kit of the invention is compatible with digital PCR amplification systems, i.e., can be used directly on a digital PCR instrument for amplification.
In a fourth aspect, there is provided a method for detecting neonatal enterovirus for non-diagnostic purposes, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
In the present invention, the sample for detection may be respiratory tract secretion 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 40-60 ℃, the time is 30min, and 1 cycle is performed; activating Taq enzyme at 95 ℃ for 1-3 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 neonatal enterovirus detection reagent, said detection comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of 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 40-60 ℃, the time is 30min, and 1 cycle is performed; activating Taq enzyme at 95 ℃ for 1-3 min for 1 cycle; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
Drawings
FIG. 1 is a graph showing the detection results of a first nucleic acid composition of the present invention (enterovirus D68, paraenterovirus A3, epstein-Barr virus 30, internal standard, respectively);
FIG. 2 is a graph of the results of the specificity of the compositions of the present invention;
FIG. 3 is a graph showing the results of single detection of the Epstein-Barr virus type 30 of the comparative example composition of the present invention;
FIG. 4 is a graph showing the results of four-joint detection 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 enterovirus D68 type probe is FAM; the fluorescent reporter group of the paraminovirus A3 probe is HEX; the fluorescent reporter group of the Epstein-Barr virus 30 type probe is CY5; the fluorescence reporter group of the internal standard probe is CY5.
Example 2 method for detecting pathogens
Fluorescent PCR amplification reaction solution: contains PCR buffer solution, taq enzyme, mg 2+, dNTP, primer, probe, etc. The specific reaction system is shown in Table 2. The enzyme mixture consists of Neoscript RT reverse transcriptase and H-Taq enzyme. H-Taq enzyme (5U/. Mu.L) and Neoscript RT enzyme (20U/. Mu.L) were mixed in a certain ratio (3. Mu. L H-Taq enzyme per person was mixed with 1. Mu. L Neoscript RT enzyme per person) (2. Mu.L per person).
TABLE 2
Reagent preparation:
The corresponding amounts of the PCR reaction solution and the enzyme mixture (38. Mu.L/part of the PCR reaction solution+2. Mu.L/part of the enzyme mixture) were taken in proportion and mixed well to obtain a PCR mixture.
Sample processing and sample addition
300. Mu.L of the sample to be tested, negative control, positive control were placed in a 1.5mL centrifuge tube, and nucleic acid extraction was performed using the nucleic acid extraction or purification reagent from St.Job's Biotechnology Co., ltd.
10 Mu L of each of the treated sample, negative control and positive control was aspirated and added to each of the reaction tubes A-B, 40 mu L of the PCR mixture was added to each of the reaction tubes, and the tube was capped.
The amplification reaction procedure of the SLAN-96P full-automatic medical PCR analysis system for PCR amplification according to a certain temperature and time setting procedure is shown in Table 3.
TABLE 3 Table 3
Result analysis and judgment:
If FAM, HEX, CY and ROX channels of the reaction system have obvious S-shaped amplification curves and Ct value is less than or equal to 36, judging positive; if FAM, HEX and ROX channels have no amplification curve (NoCt) or Ct > 36, then the result is negative, as shown in Table 4.
TABLE 4 Table 4
Example 3 detection results of test samples of the inventive composition
The primers and probes shown in the example 1 are used for carrying out PCR detection on a macrostone fluorescence quantitative PCR instrument on a mixed sample of enterovirus D68 type, paraenterovirus A3 type and Epstein-Barr virus 30 type according to the method of the example 2, and the detection result is shown in the figure 1, so that the composition can well detect various pathogens.
Example 4 sensitivity of the composition of the invention
Using the composition of example 1 of the present invention, LOD (sensitivity) detection was performed on each target to simulate a clinical sample, and multiplex PCR detection was performed on a macrostone fluorescent quantitative PCR instrument. The results are shown in Table 5, which demonstrate that each channel can still be accurately detected for samples as low as 500 copies/mL, indicating a sensitivity of 500 copies/mL for the compositions of the present invention.
TABLE 5
EXAMPLE 5 specificity of the composition of the invention
In order to test the specificity of the composition in example 1 of the present invention, the specificity experiment shows that the method of the present invention has no cross reaction to other neonatal enteroviruses (enterococcus faecium, staphylococcus aureus, salmonella typhi, salmonella paratyphi a/b/c, salmonella enteritidis, salmonella typhimurium, yersinia small intestine, shigella dysenteriae, campylobacter, diarrheagenic escherichia coli, clostridium difficile, clostridium perfringens, helicobacter pylori, neonatal enterovirus type 71, astrovirus, such as virus, norovirus, rotavirus, enteroadenovirus) and the like, and the result of the specificity experiment is shown in fig. 2. The results show that the method of the invention has no cross-reaction to the enteric pathogens.
EXAMPLE 6 interference resistance of the composition of the invention
The test results show that the potential PCR inhibitors/interferences such as dexamethasone (50 mug/mL), cefmenoxime hydrochloride (50 mug/mL), zanamivir (100 mug/mL), ribavirin (100 mug/mL), clindamycin (50 mug/mL), norfloxacin (25 mug/mL), tobramycin (50 mug/mL), heme (10 mug/mL) and hemoglobin (10%) have no obvious influence on the test kit, and the amplification results of the PCR reaction liquid in the presence of infectious substances are shown in the following table 6.
TABLE 6
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 inventor also designs other primers and probes to form different detection systems (sequences are not shown), and the primers and probes are also used for detecting the pathogens, and specific detection results are shown in fig. 3 and 4, the primer probes of the comparative example can be effectively detected in a single-detection Epstein-Barr virus 30 type system, and the omission detection exists when the comparative example is combined for detection.
Claims (10)
1. A composition for neonatal enterovirus detection, comprising:
an upstream primer, a downstream primer and a probe for detecting enterovirus D68 as shown in SEQ ID NO. 1-3;
An upstream primer, a downstream primer and a probe for detecting the Paracolone virus A3 type as shown in SEQ ID NO. 4-6; and
The upstream primer, the downstream primer and the probe for detecting the type 30 of the Epstein-Barr virus are shown in SEQ ID NOs 7 to 9.
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.10 to 12.
3. The composition of claim 2, wherein the fluorophores of the composition probes 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 the enterovirus D68 type probe is FAM; the fluorescent reporter group of the paraminovirus A3 probe is HEX; the fluorescent reporter group of the Epstein-Barr virus 30 type probe is CY5; the fluorescence reporter group of the internal standard 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 neonatal enterovirus.
7. A kit for detecting neonatal enterovirus, the 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: at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, a reverse transcriptase, a DNA polymerase, dNTP, dUTP, UNG enzyme, a PCR buffer, and Mg 2+.
10. Use of a composition for the preparation of a reagent for neonatal enterovirus detection, said detection comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of any one of claims 1 to 5 or the kit of any one of claims 7 to 9;
3) The results were obtained and analyzed.
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