CN114410842A - Nucleic acid detection kit, primer probe set and detection method for four enteroviruses - Google Patents
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Abstract
The invention provides a nucleic acid detection kit, a primer probe set and a detection method for four enteroviruses, belonging to the technical field of genetic engineering.A forward primer of an enterovirus EV is a sequence EV-F1 or EV-F2; the downstream primer is a sequence EV-R, the specific probe is a sequence EV-P, and the upstream primer of the EV71 is a sequence EV 71-F; the downstream primer is a sequence EV71-R, the specific probe is a sequence EV71-P, and the upstream primer of CA16 is a sequence CA 16-F; the downstream primer is a sequence CA16-R, the specific probe is a sequence CA16-P, and the upstream primer of CA6 is a sequence CA 6-F; the downstream primer is a sequence CA6-R, the specific probes are sequences CA6-P1 and CA6-P2, and the detection kit disclosed by the invention is used for detecting by one-step fluorescent quantitative PCR (polymerase chain reaction), is simple and convenient to operate and can quickly read results.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a nucleic acid detection kit, a primer probe set and a detection method for four enteroviruses.
Background
The enteroviridae belong to the genus enteroviruses of the family enteropicornaviridae and are conventionally classified into Polioviruses (PV), echoviruses (Echo), coxsackieviruses A, B groups (coxsackievirussa, B, CVA, B) and novel enteroviruses. In the latest enterovirus classification, the international committee for virus classification (ICTV) divides it into 4 groups, A, B, C, D groups for enteroviruses, respectively, based on biological and genetic characteristics. More than one hundred enterovirus serotypes have been reported to date.
Hand-foot-and-mouth disease is an acute infectious disease caused by various enteroviruses (most common in Coxsackie group A16 type and enterovirus 71 type). It is mostly seen in preschool children, and is characterized by fever, rash in hands, feet, oral cavity, etc., herpes or herpangina. In a few severe cases (mostly caused by EV71 infection), severe complications such as aseptic meningitis, brainstem encephalitis, neurogenic pulmonary edema, acute flaccid paralysis and the like can occur, and high fatality rate and disability rate are caused, so that the health and life safety of children are seriously harmed. Children patients and recessive infected persons are main infectious sources, and the recessive infection rate of the hand-foot-and-mouth disease is high. The disease can occur all the year round, but is common in summer and autumn, and mainly occurs in 4-9 months. The disease can be developed at any age, and the incidence rate is highest in the age group below 3 years old. The disease has strong infectivity, is easy to cause epidemic, and is generally easy to feel for infants and children.
At present, enterovirus detection methods mainly comprise three types: virus isolation culture, immunological methods, and molecular biological methods. Virus isolation and culture are the gold standard for viral disease diagnosis, but the operation is complicated, the detection time is long, and the positive rate is low. The immunology method is the main method of traditional typing, but the source of antiserum is very limited, and the requirement of clinical pathogen diagnosis cannot be met. Molecular biology methods based on related technologies such as PCR have been greatly developed due to their advantages of sensitivity, rapidity, and the like. The multiplex real-time fluorescence quantitative PCR technology overcomes the defects of the prior clinical diagnosis by virtue of the advantages of full-closed single-tube amplification, simplicity, rapidness, good repeatability, real-time quantification, less pollution and the like, has high specificity and sensitivity, provides accurate and reliable laboratory basis for the clinical diagnosis, and can be used as an effective and rapid detection means for the clinical pathogen diagnosis.
Disclosure of Invention
The invention aims to provide a nucleic acid detection kit, a primer probe set and a detection method for four enteroviruses, which can simultaneously detect whether the enteroviruses exist in a stool sample through PCR reaction, carry out typing identification on main enterovirus types, are more convenient and quicker than a single fluorescent quantitative PCR method and save cost.
The technical scheme of the invention is realized as follows:
the invention provides a primer probe set for enteroviruses EV, EV71, CA16 and CA6, wherein an upstream primer of the enterovirus EV is a sequence EV-F1 or EV-F2; the downstream primer is a sequence EV-R, the specific probe is a sequence EV-P, and the nucleotide sequences of the upstream primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3; the nucleotide sequence of the specific probe is shown as SEQ ID NO. 4;
the upstream primer of the EV71 has a sequence EV 71-F; the downstream primer is a sequence EV71-R, the specific probe is a sequence EV71-P, and the nucleotide sequences of the upstream primer are respectively shown as SEQ ID NO. 5; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6; the nucleotide sequence of the specific probe is shown as SEQ ID NO. 7;
the upstream primer of CA16 is sequence CA 16-F; the downstream primer is a sequence CA16-R, the specific probe is a sequence CA16-P, and the nucleotide sequences of the upstream primer are respectively shown as SEQ ID NO. 8; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 9; the nucleotide sequence of the specific probe is shown as SEQ ID NO. 10;
the upstream primer of CA6 is sequence CA 6-F; the downstream primer is a sequence CA6-R, the specific probes are sequences CA6-P1 and CA6-P2, wherein the nucleotide sequences of the upstream primer are respectively shown as SEQ ID NO. 11; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 12; the nucleotide sequence of the specific probe is shown as SEQ ID NO. 13 and SEQ ID NO. 14.
As a further improvement of the invention, the kit comprises the primer probe set.
As a further improvement of the invention, the 5 'end of the specific probe sequence of the enterovirus EV is modified by ROX, and the 3' end is modified by BHQ 2; the 5 'end of the specific probe sequence of the EV71 is modified by CY5, and the 3' end of the specific probe sequence of the EV71 is modified by BHQ 2; the 5 'end of the specific probe sequence 9 of the CA16 is modified by JOE, and the 3' end is modified by BHQ 1; the 5 'end of the specific probe sequence of the CA6 is modified by FAM, and the 3' end is modified by BHQ 1.
The invention is further improved by comprising a fluorescent PCR detection reaction solution, the primer probe group mixed solution, an enzyme mixed solution, a positive control substance and a negative control substance.
As a further improvement of the invention, the forward primers of enteroviruses EV, EV71, CA16 and CA6 are as follows: reverse primer: the molar weight of the specific fluorescent probe is as follows:
EV：200nm:200nm:160nm；
EV71：400nm:200nm:600nm；
CA16：200nm:300nm:600nm；
CA6：200nm:200nm:60um:60nm。
as a further improvement of the invention, the components are as follows:
(1) RT-PCR reaction solution: comprises fluorescent PCR buffer solution, dNTP and DEPC water;
(2) primer probe mixed solution: including the primer probe set described above;
(3) mixing enzyme solution: including RNase inhibitors, reverse transcriptase, DNA polymerase;
(4) positive control: EV, EV71, CA16 and CA6 source RNA of the amplified target segment containing the primer probe group;
(5) negative control: the TE solution was obtained.
As a further improvement of the invention, the kit is stored at-20 ℃ in the absence of light.
The invention further provides a quadruple fluorescence PCR method, which comprises the following steps:
s1, extracting total RNA of a sample to be detected;
s2, performing recombinase polymerase amplification reaction by using the total RNA as a template and using the primer probe group;
s3, collecting fluorescence signals of FAM, JOE, CY5 and ROX detection channels to obtain a detection result:
s301, quality control standard: fluorescence indexes of positive control FAM, JOE, CY5 and ROX fluorescence signals are increased, and Ct value is less than or equal to 30; the negative control and the blank control have no fluorescence index increase, and the Ct value is more than or equal to 40;
s302, positive sample: when the step S301 is established, the fluorescence index of FAM or/and JOE or/and CY5 or/and ROX is increased, and the Ct value is less than or equal to 37, which indicates that the sample is detected to be common to CA6 or/and CA16 or/and EV71 or/and enterovirus;
s303, negative sample: when the step S301 is established, FAM or/and JOE or/and CY5 or/and ROX do not have fluorescence exponential increase, and the Ct value is more than or equal to 40, which indicates that the sample is not detected to be universal to CA6 or/and CA16 or/and EV71 or/and enterovirus;
s304, suspected sample: when the step S301 is established, the fluorescence index of FAM or/and JOE or/and CY5 or/and ROX is increased, and the Ct value is more than or equal to 37 and less than or equal to 40, and the detection is repeated once; if the Ct value of the recheck result is still more than or equal to 37 and less than or equal to 40 and has obvious exponential increase, the result is judged to be positive, otherwise, the result is negative.
As a further improvement of the present invention, the reaction solution is prepared as shown in table 1 below:
TABLE 1
As a further improvement of the present invention, the reaction conditions are as shown in table 2 below:
TABLE 2
The invention has the following beneficial effects: the invention develops a quadruple real-time fluorescent quantitative RT-PCR detection kit for detecting enteroviruses EV and three subtypes (EV71, CVAl6 and CVA6) thereof by using a one-step real-time fluorescent quantitative RT-PCR technology and adopting highly specific primers and fluorescent labeled probes of EV, EV71, CVAl6 and CVA6 viruses. The invention can simultaneously detect whether the enterovirus exists in the excrement sample through a PCR reaction, and simultaneously carry out typing identification on the types of the main enteroviruses, thereby being more convenient and rapid and saving the cost compared with a single fluorescent quantitative PCR method. Meanwhile, the detected virus is accurately quantified in real time, and according to the titer of virus infection, early diagnosis is provided for clinical patients, and a reference basis is provided for formulation of a clinical treatment scheme; can be applied to laboratory emergency diagnosis of outbreak epidemic caused by enteroviruses, rapid screening and typing of the enteroviruses, clinical diagnosis and the research of epidemiology of hand-foot-and-mouth disease.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph showing the amplification of a CA6 virus-positive specimen in example 1;
FIG. 2 is a graph showing the amplification of a CA16 virus-positive specimen in example 1;
FIG. 3 is a graph showing the amplification of the EV71 virus-positive specimen in example 1;
FIG. 4 is a graph showing the amplification of other enterovirus-positive specimens in example 1;
FIG. 5 is a graph showing the amplification curve of the positive control of the kit;
FIG. 6 is a graph showing the amplification curve of the negative control of the kit.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 self-made samples
Virus samples containing EV, EV71, CA16 and CA6 were extracted and detected using an externally purchased nucleic acid extraction kit (major nucleic acid extraction kit). The primer solution, the probe solution and the positive control are the main components of the kit, and the negative control is a TE solution. The configuration is carried out according to the table 3, and the relevant parameters are set according to the table 4 for on-machine detection (ABI fluorescence quantitative PCR instrument, model 7500).
TABLE 3 reaction System
| Reagent | 25 μ L System |
| RT-PCR reaction solution | 17μL |
| Primer probe mixed solution | 2μL |
| Mixed enzyme solution | 1μL |
| Sample (I) | 5μL |
TABLE 4 reaction conditions
The results are shown in Table 5:
quality control standard: the fluorescence log of positive control FAM, JOE, Cy5 and ROX fluorescence signals increases, and Ct value is less than or equal to 30.
Detecting a sample: FAM, JOE, Cy5 and ROX all have fluorescence logarithm increase, and Ct value is less than or equal to 35.0, which indicates that EV, EV71, CA16 and CA6 are detected by the kit respectively.
③ negative control: the results that FAM, JOE, Cy5 and ROX have no fluorescence logarithmic increase and Ct value is more than or equal to 40.0 show the specificity and anti-interference of the method.
TABLE 5 results of the experiment
And (4) conclusion:
in example 1, amplification was performed using a fluorescent quantitative PCR instrument (ABI-7500) to obtain a graph: from the amplification curves (FIGS. 1-6) we can derive:
1. the positive control, FAM, JOE, Cy5 and ROX channels, showed a logarithmic increase in fluorescence, wherein the FAM showed a CT value of 24, JOE showed a CT value of 24, CY5 showed a CT value of 24, and ROX showed a CT value of 24, and the positive control showed a normal result (FIG. 5).
2, the FAM channel of the CA6 positive virus specimen has the fluorescence logarithmic increase, the CT value is 28, the ROX channel has the fluorescence logarithmic increase, and the CT value is 28; the JOE and Cy5 channels showed no log increase in fluorescence, and the detection results were consistent with the type of virus identified (FIG. 1).
3, the JOE channel of the CA16 positive virus specimen has the logarithmic increase of fluorescence, the CT value is 32, the ROX channel has the logarithmic increase of fluorescence, and the CT value is 32; the FAM and Cy5 channels have no increase of fluorescence logarithm, and the detection result is consistent with the verified virus species (FIG. 2).
4, the Cy5 channel of the EV71 positive virus specimen has the fluorescence logarithmic growth, the CT value is 23, the ROX channel has the fluorescence logarithmic growth, and the CT value is 23; FAM and JOE channels did not show a logarithmic increase in fluorescence, and the detection results were consistent with the verified virus species (FIG. 3).
5. The ROX channels of other enterovirus positive virus specimens have fluorescence logarithm increase, and the CT value is 29; the FAM, JOE, Cy5 channels did not exhibit log increase in fluorescence, and the results were consistent with the type of virus identified (FIG. 4).
6. Negative controls, such as FAM, JOE, Cy5, and ROX channels, all showed no increase in fluorescence log, and the negative control was normal (FIG. 6).
The specificity and interference resistance of the method of the application are shown.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Guangzhou Shengling medical science and technology Co., Ltd
<120> nucleic acid detection kit, primer probe set and detection method for four enteroviruses
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Claims (10)
1. Primer probe sets of enteroviruses EV, EV71, CA16 and CA6, which are characterized in that:
wherein, the upstream primer of the EV is sequences EV-F1 and EV-F2; the downstream primer is a sequence EV-R, the specific probe is a sequence EV-P, and the nucleotide sequences of the upstream primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3; the nucleotide sequence of the specific probe is shown as SEQ ID NO. 4;
the upstream primer of the EV71 has a sequence EV 71-F; the downstream primer is a sequence EV71-R, the specific probe is a sequence EV71-P, and the nucleotide sequences of the upstream primer are respectively shown as SEQ ID NO. 5; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6; the nucleotide sequence of the specific probe is shown as SEQ ID NO. 7;
the upstream primer of CA16 is sequence CA 16-F; the downstream primer is a sequence CA16-R, the specific probe is a sequence CA16-P, and the nucleotide sequences of the upstream primer are respectively shown as SEQ ID NO. 8; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 9; the nucleotide sequence of the specific probe is shown as SEQ ID NO. 10;
the upstream primer of CA6 is sequence CA 6-F; the downstream primer is a sequence CA6-R, the specific probes are sequences CA6-P1 and CA6-P2, wherein the nucleotide sequences of the upstream primer are respectively shown as SEQ ID NO. 11; the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 12; the nucleotide sequence of the specific probe is shown as SEQ ID NO. 13 and SEQ ID NO. 14.
2. The quadruple nucleic acid detection kit for enteroviruses EV, EV71, CA16 and CA6, which comprises the primer probe set according to claim 1.
3. The quadruple nucleic acid detection kit for enteroviruses EV, EV71, CA16 and CA6 as claimed in claim 2, wherein the 5 'end of the specific probe sequence for enteroviruses EV is modified with ROX, and the 3' end is modified with BHQ 2; the 5 'end of the specific probe sequence of the EV71 is modified by CY5, and the 3' end of the specific probe sequence of the EV71 is modified by BHQ 2; the 5 'end of the specific probe sequence 9 of the CA16 is modified by JOE, and the 3' end is modified by BHQ 1; the 5 'end of the specific probe sequence of the CA6 is modified by FAM, and the 3' end is modified by BHQ 1.
4. The quadruple nucleic acid detection kit for enteroviruses EV, EV71, CA16 and CA6 according to claim 2, which comprises a fluorescent PCR detection reaction solution, the primer probe set mixture according to claim 1, an enzyme mixture, a positive control and a negative control.
5. The quadruple nucleic acid detection kit for enteroviruses EV, EV71, CA16 and CA6 according to claim 4, which comprises the following components:
(1) RT-PCR reaction solution: comprises fluorescent PCR buffer solution, dNTP and DEPC water;
(2) primer probe mixed solution: comprising the primer probe set of claim 1;
(3) mixing enzyme solution: including RNase inhibitors, reverse transcriptase, DNA polymerase;
(4) positive control: EV, EV71, CA16 and CA6 derived RNA of an amplified target fragment containing the primer probe set of claim 1;
(5) negative control: the TE solution was obtained.
6. The quadruple nucleic acid detection kit for enteroviruses EV, EV71, CA16 and CA6 according to claim 2, wherein the kit is stored at-20 ℃ in the absence of light.
7. The quadruple nucleic acid detection kit for enteroviruses EV, EV71, CA16 and CA6 of claim 2, wherein the molar ratio of the forward primer to the reverse primer to the specific fluorescent probe is as follows:
EV：200：200：160；
EV71：400：200：600；
CA16：200：300：600；
CA6：200：200：60：60。
8. a quadruple fluorescent PCR method is characterized by comprising the following steps:
s1, extracting total RNA of a sample to be detected;
s2, using the total RNA as a template and the primer probe group of claim 1 to perform recombinase polymerase amplification reaction;
s3, collecting fluorescence signals of FAM, JOE, CY5 and ROX detection channels to obtain a detection result:
s301, quality control standard: fluorescence indexes of positive control FAM, JOE, CY5 and ROX fluorescence signals are increased, and Ct value is less than or equal to 30; the negative control and the blank control have no fluorescence index increase, and the Ct value is more than or equal to 40;
s302, positive sample: when the step S301 is established, the fluorescence index of FAM or/and JOE or/and CY5 or/and ROX is increased, and the Ct value is less than or equal to 37, which indicates that the sample is detected to be common to CA6 or/and CA16 or/and EV71 or/and enterovirus;
s303, negative sample: when the step S301 is established, FAM or/and JOE or/and CY5 or/and ROX do not have fluorescence exponential increase, and the Ct value is more than or equal to 40, which indicates that the sample is not detected to be universal to CA6 or/and CA16 or/and EV71 or/and enterovirus;
s304, suspected sample: when the step S301 is established, the fluorescence index of FAM or/and JOE or/and CY5 or/and ROX is increased, and the Ct value is more than or equal to 37 and less than or equal to 40, and the detection is repeated once; if the Ct value of the recheck result is still more than or equal to 37 and less than or equal to 40 and has obvious exponential increase, the result is judged to be positive, otherwise, the result is negative.
9. The quadruple fluorescent PCR method according to claim 8, wherein the reaction solution is prepared as shown in the following Table 1:
TABLE 1
10. The quadruple fluorescent PCR method according to claim 8, wherein the reaction conditions are as shown in Table 2 below:
TABLE 2
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851391A (en) * | 2012-07-20 | 2013-01-02 | 宁波检验检疫科学技术研究院 | Human enterovirus four-color fluorescence RT-PCR detection kit and detection method |
| CN107513584A (en) * | 2017-09-25 | 2017-12-26 | 浙江大学 | A kind of five heavy fluorescence quantitative kits for detecting enterovirus |
| CN111748648A (en) * | 2019-12-24 | 2020-10-09 | 深圳市人民医院 | Kit for detecting five infant hand-foot-and-mouth disease series viruses and application thereof |
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- 2022-01-27 CN CN202210098940.3A patent/CN114410842A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851391A (en) * | 2012-07-20 | 2013-01-02 | 宁波检验检疫科学技术研究院 | Human enterovirus four-color fluorescence RT-PCR detection kit and detection method |
| CN107513584A (en) * | 2017-09-25 | 2017-12-26 | 浙江大学 | A kind of five heavy fluorescence quantitative kits for detecting enterovirus |
| CN111748648A (en) * | 2019-12-24 | 2020-10-09 | 深圳市人民医院 | Kit for detecting five infant hand-foot-and-mouth disease series viruses and application thereof |
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