CN110863065A - Kit for detecting four enteroviruses and detection method thereof - Google Patents
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Abstract
The invention discloses a kit for detecting four enteroviruses and a detection method thereof. Relates to a coxsackievirus A10 type, a coxsackievirus A9 type, a coxsackievirus B2 type and a B5 type. The invention applies a one-step real-time fluorescent quantitative RT-PCR technology, adopts highly specific primers and fluorescent labeled probes of CVA10, CVA9, CVB2 and CVB5 viruses, can judge whether the 4 types of coxsackie viruses exist in a sample to be detected through a PCR reaction, and is more convenient and quicker than a single fluorescent quantitative PCR method and saves cost. The invention can accurately quantify the detected virus in real time, provide a tool for clinical early diagnosis according to the titer of virus infection, and provide a reference basis for the 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.
Description
Technical Field
The invention belongs to the technical field of virus detection kits, relates to a real-time fluorescence RT-PCR detection kit, and particularly relates to a kit for simultaneously detecting four types of coxsackie virus strains and a detection method thereof.
Background
Coxsackieviruses (coxsackieviruses) are enteroviruses (enteroviruses) which are divided into A and B types, are common viruses infecting human bodies through respiratory tracts and digestive tracts, and can cause cold symptoms such as fever, sneeze, cough and the like after infection, and are also main pathogens of hand-foot-and-mouth diseases. Hand-foot-and-mouth disease (HFMD) is a common infectious disease caused by human enterovirus infection, and has skin rash symptoms on hands, feet, mouths and buttocks as main clinical symptoms, mainly mild symptoms, but severe cases and death cases are reported. According to the latest data of infectious disease morbidity and mortality statistics reported nationwide by the China center for disease prevention and control (http:// www.chinacdc.cn/tjsj/fdcrbbg /), 15,432,764 cases of hand-foot-and-mouth diseases are totally contained in the China continent from 2008 to 2016 (month 7), and 3,486 deaths are contained. The infection situation of the hand-foot-mouth disease in China is still severe.
In most reports, the pathogens causing hand-foot-and-mouth disease are enterovirus 71 (EV71) and coxsackievirus A16 (Coxsackie virus A16 and CVA 16). In recent years, the number of hand-foot-and-mouth disease cases and epidemic events caused by enteroviruses other than EV71 and CVA16 is increasing worldwide. Infection of singapore CVA6 and CVA10 in 2008 causes 35.3% of cases of hand-foot-and-mouth disease; . In 2012, CV-B2 was associated with severe hand-foot-and-mouth disease in an epidemic situation of hands, feet and mouths, which was developed in Yunnan province; in 2010-2012, the foot-mouth epidemic situation in Shijiazhuang area has a prevalence of CV-B3. Enterovirus types such as Echovuises 6 (E-6), E-9, E-30 and CV-A5 have been reported. Therefore, the types of human enteroviruses causing hand-foot-and-mouth disease are many, and the pathogenic spectrum is easy to generate temporal and spatial transition.
At present, in the enterovirus molecular diagnosis market in China, the detection of the universal enterovirus or the enterovirus 71 and the Coxsackie virus A16 is mainly used, so that the detection of pathogens of hand-foot-and-mouth diseases is unclear or missed, the treatment delay of patients, the waste of diagnostic reagents and the like are caused. The coxsackieviruses now under major investigation, coxsackievirus group a type 9 (coxsackievirus a9, CVA9) and coxsackievirus group a type 10 (coxsackievirus a10, CVA10), coxsackievirus group B type 2 (coxsackievirus B2, CVB2) and coxsackievirus group B type 5 (coxsackievirus B5, CVB5) are also receiving increasing attention.
In order to better guide the research on the four viruses, a product capable of detecting coxsackievirus A9, coxsackievirus A10, coxsackievirus B2 and coxsackievirus B5 needs to be developed. However, the conventional detection method or detection product can only detect one virus type at a time, and when multiple virus types are detected at one time, the multiple primers interfere with each other, which causes problems of primer dimer, false positive, etc., and is not favorable for the research of the four viruses.
Disclosure of Invention
In order to solve the problem that various types of coxsackie viruses cannot be detected simultaneously in the prior art, the invention provides a detection primer and a probe of 4 types of coxsackie virus nucleic acid based on a fluorescent quantitative PCR technology, relating to coxsackie virus A10 type, coxsackie virus A9 type, coxsackie virus B2 type and B5 type, so as to realize the rapid, effective and accurate detection of coxsackie virus types.
The invention also aims to provide a quadruple fluorescent quantitative kit for detecting the four types of coxsackie viruses, which is a one-step quadruple real-time fluorescent quantitative RT-PCR detection kit.
The invention further aims to provide application of the kit in preparing a reagent for detecting the 4 types of coxsackieviruses.
The fourth purpose of the invention is to provide a using method of the kit.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, a detection primer and a probe for 4 types of coxsackie virus nucleic acids are provided, the detection primer and the probe comprise specific amplification primer sequences of 4 types of genes and specific fluorescent probe sequences corresponding to the primer, a fluorescent reporter group is marked at the 5 'end of the probe sequence, a fluorescent quenching group is marked at the 3' end of the probe sequence, and the detection primer and the probe sequences of each type are as follows:
coxsackievirus type a 9:
an upstream primer CVA 9-F: the nucleotide sequence is shown as SEQ ID NO.1,
the nucleotide sequence of the downstream primer CVA9-R is shown as SEQ ID NO 2,
the nucleotide sequence of the specific probe CVA9-P is shown as SEQ ID NO 3,
coxsackievirus type a 10:
an upstream primer CVA 10-F: the nucleotide sequence is shown as SEQ ID NO.4,
the nucleotide sequence of the downstream primer CVA10-R is shown as SEQ ID NO.5,
the nucleotide sequence of the specific probe CVA10-P is shown as SEQ ID NO 6,
coxsackievirus B2 type:
an upstream primer CVB 2-F: the nucleotide sequence is shown as SEQ ID NO.7,
the nucleotide sequence of the downstream primer CVB2-R is shown as SEQ ID NO 8,
the nucleotide sequence of the specific probe CVB2-P is shown as SEQ ID NO 9,
coxsackievirus B5 type:
an upstream primer CVB 5-F: the nucleotide sequence is shown as SEQ ID NO.10,
the nucleotide sequence of the downstream primer CVB5-R is shown as SEQ ID NO.11,
the nucleotide sequence of the specific probe CVB5-P is shown as SEQ ID NO. 12.
cva9-f：5'-ccaaccacggagcaggtga-3’(seq id no:1),
cva9-r:5'-gaaacacggacacccaaagtagt-3'(seq id no:2),
cva9-p:5'-caacccagcaactagcctgtcgtaacgc-3'(seq id no:3),
cva10-f：5'-ccgttgtgtggttaacagaaa-3'(seq id no:4),
cva10-r:5'-gtccccccatctgtgaggtt-3'(seq id no:5),
cva10-p:5'-tatcaaccacttcttctcccgct-3'(seq id no:6),
cvb2-f：5'-gcccctgaatgcggc-3'(seq id no:7),
cvb2-r:5'-aattgtcaccataagcagc-3'(seq id no:8),
cvb2-p:5'-cggaaccgactactttgggtgtccgt-3'(seq id no:9),
cvb5-f：5'-acatggtgcgaagagtctattgag-3'(seq id no:10),
cvb5-r:5'-tgctccgcagttaggattagc-3'(seq id no:11),
cvb5-p:5'-ttggtagtcctccggcccctgaat-3'(seq id no:12)。
Preferably, the fluorescent reporter group carried on the above-mentioned detection probe of coxsackievirus type a9, detection probe of coxsackievirus type a10, detection probe of coxsackievirus type B2 or detection probe of coxsackievirus type B5 is selected from 6-carboxyfluorescein (6-carboxyfluorescein, 6-FAM), Hexachloro-6-methylfluorescein (Hexachloro-6-methylfluorescein, HEX), VIC fluorescent dye, tetrachloro-6-carboxyfluorescein (tetrachloro-6-carboxyfluorescein, TET), Carboxy-X-rhodamine, 6-carboxytetramethylrhodamine (6-carboxytetramethylrhodamine, TAMRA), sulforhodamine (S μ Lforhodamine 101, Texas Red), 6-Carboxy-4 ', 5 ' -dichloro-2 ', 7 ' -dimethoxyfluorescein succinimide ester (6-carboxyimide, 4 ', at least one of 5 ' -dichoro-2 ', 7 ' -dimethoxfluorescein, JOE), cyanine 3(cyanine3, Cy3), cyanine3.5 (cyanine3.5, Cy3.5), cyanine 5(cyanine5, Cy5), and cyanine5.5 (cyanine5.5, Cy 5.5); the fluorescence quenching group on the detection probe of the coxsackie virus A9 type, the detection probe of the coxsackie virus A10 type, the detection probe of the coxsackie virus B2 type and the detection probe of the coxsackie virus B5 type is at least one selected from 6-carboxytetramethylrhodamine, 4- (4-dimethylamino phenylazo) benzoic acid, a Black Hole Quencher 1(Black Hole Quencher 1 and BHQ1), a Black Hole Quencher 2(Black Hole Quencher 2 and BHQ2) or a Black Hole Quencher 3(Black Hole Quencher 3 and BHQ 3).
Preferably, the fluorescent reporter group is carried on the detection probe of coxsackie virus A9, the detection probe of coxsackie virus A10, the detection probe of coxsackie virus B2 or the detection probe of coxsackie virus B5, and is selected from one of 6-carboxyfluorescein (6-carboxyfluorescein, 6-FAM), VIC fluorescent dye, sulforhodamine (S mu Lforhodamine 101, Texas Red) and sulforhodamine (S mu Lforhodamine 101, Texas Red); the fluorescence quenching group is selected from one of Black Hole Quencher 1(Black Hole Quencher 1, BHQ1) or Black Hole Quencher 2(Black Hole Quencher 2, BHQ 2).
Most preferably, the fluorescent reporter group of the coxsackievirus A9 type detection probe is 6-carboxyfluorescein (6-carboxyfluorescein, 6-FAM), and the fluorescent quencher group is BHQ1; the fluorescent reporter group of the Coxsackie virus A10 type detection probe is VIC fluorescent dye, and the fluorescent quenching group is BHQ1; the fluorescence reporter group of the Coxsackie virus B2 type detection probe is sulforhodamine (S mu Lforhodamine 101, Texas Red), and the fluorescence quenching group is BHQ2; the fluorescent reporter group of the detection probe of the coxsackievirus B5 type is sulforhodamine (S mu Lforhodamine 101, Texas Red), and the fluorescent quenching group is BHQ 2.
In a second aspect, the invention provides a kit for simultaneously detecting 4 types of coxsackieviruses, which comprises any one of the detection primers and the detection probes for the 4 types of coxsackieviruses, wherein the detection probe for the coxsackievirus A9, the detection probe for the coxsackievirus A10, the detection probe for the coxsackievirus B2 and the detection probe for the coxsackievirus B5 are respectively provided with different fluorescent reporter groups.
Preferably, the kit for simultaneously detecting 4 types of coxsackie viruses is used for preparing a primer and probe mixed solution according to the following proportion: the molar ratio of the amplification primer pair of the coxsackievirus A9 to the detection probe of the coxsackievirus A9 is 5:1, the molar ratio of the amplification primer pair of the coxsackievirus A10 to the detection probe of the coxsackievirus A10 is 5:2, the molar ratio of the amplification primer pair of the coxsackievirus B2 to the detection probe of the coxsackievirus B2 is 2:1, the molar ratio of the amplification primer pair of the coxsackievirus B5 to the detection probe of the coxsackievirus B5 is 10:3, and the molar ratio of the detection probe of the coxsackievirus A9, the detection probe of the coxsackievirus A10, the detection probe of the coxsackievirus B2 and the detection probe of the coxsackievirus B5 is 2:4:5: 6.
Preferably, the kit for simultaneously detecting 4 types of coxsackieviruses further comprises a positive quality control product and a negative quality control product, wherein the negative quality control product is a physiological saline solution, and the positive control product comprises RNA formed by reverse transcription of a plasmid containing a coxsackievirus A9 type target sequence (named as a coxsackievirus A9 type positive standard), RNA formed by reverse transcription of a plasmid containing a coxsackievirus A10 type target sequence (named as a coxsackievirus A10 type positive standard), RNA formed by reverse transcription of a plasmid containing a coxsackievirus B2 type target sequence (named as a coxsackievirus B2 type positive standard) and RNA formed by reverse transcription of a plasmid containing a coxsackievirus B5 type target sequence (named as a coxsackievirus B5 type positive standard).
Specifically, the preparation process of the positive quality control product is as follows: respectively extracting RNAs of a coxsackievirus A9 type, a coxsackievirus A10 type, a coxsackievirus B2 type and a coxsackievirus B5 type as reverse transcription PCR templates, and respectively amplifying corresponding target sequences by using corresponding primers; connecting the amplified product into a vector to respectively obtain a plasmid containing a coxsackievirus A9 type target sequence, a plasmid containing a coxsackievirus A10 type target sequence, a plasmid containing a coxsackievirus B2 type target sequence and a plasmid containing a coxsackievirus B5 type target sequence; the plasmids are respectively subjected to reverse transcription to obtain corresponding RNA, namely RNA formed by reverse transcription of a plasmid containing a coxsackievirus A9 type target sequence, RNA formed by reverse transcription of a plasmid containing a coxsackievirus A10 type target sequence, RNA formed by reverse transcription of a plasmid containing a coxsackievirus B2 type target sequence and RNA formed by reverse transcription of a plasmid containing a coxsackievirus B5 type target sequence, wherein the vector is pUC57-Kan vector.
In one specific example, the positive quality control is prepared as follows: respectively taking the synthetic fragment of CA9, the synthetic fragment of CA10, the synthetic fragment of CB2 and the Coxsackie synthetic fragment of B5 as templates, and respectively amplifying corresponding amplified fragments by using corresponding primers; connecting the amplified products into a vector to respectively obtain a plasmid containing a coxsackievirus A9 type amplified fragment, a plasmid containing a coxsackievirus A10 type amplified fragment, a plasmid containing a coxsackievirus B2 type amplified fragment and a plasmid containing a coxsackievirus B5 type amplified fragment; and respectively carrying out reverse transcription on the plasmids to obtain corresponding RNA, namely a coxsackievirus A9 type positive quality control product, a coxsackievirus A10 type positive quality control product, a coxsackievirus B2 type positive quality control product and a coxsackievirus B5 type positive quality control product.
In one embodiment, the negative quality control product is a physiological saline solution that does not contain coxsackievirus A9, coxsackievirus A10, coxsackievirus B2 and coxsackievirus B5. The negative quality control material may be a physiological saline solution not containing the coxsackievirus A9 type gene, the coxsackievirus A10 type gene, the coxsackievirus B2 type gene and the coxsackievirus B5 type gene.
Preferably, the kit for simultaneously detecting 4 types of Coxsackie viruses further comprises at least one of an RNA extraction reagent, dNTPs, a PCR reaction solution and an enzyme mixed solution, wherein the PCR reaction solution comprises 220 mM-280 mM Tris-base, 0.2% -0.3% TritonX-100 in percentage by mass and 20 mmol/L-30 mmol/L MgCl2(ii) a The enzyme mixed solution comprises Taq enzyme and reverse transcriptase.
More preferably, in the kit for simultaneously detecting 4 types of Coxsackie viruses, the PCR reaction solution comprises 250mM Tris-base, 0.25% TritonX-100 in percentage by mass and 25mmol/L MgCl2。
More preferably, in the kit for simultaneously detecting 4 types of coxsackieviruses, the Taq enzyme in the enzyme mixed solution is hot-start Taq enzyme, the reverse transcriptase is MMLV reverse transcriptase, and the volume ratio of the hot-start Taq enzyme to the reverse transcriptase is 4: 1.
More preferably, in the kit for simultaneously detecting 4 types of coxsackie viruses, the enzyme mixture further comprises an RNase inhibitor, and the volume ratio of the RNase inhibitor to the Taq enzyme is 3: 4.
In a third aspect, the application of the kit for simultaneously detecting the 4 types of coxsackieviruses in preparing a reagent for detecting the 4 types of coxsackieviruses is provided.
In a fourth aspect, there is provided a method for simultaneously detecting 4 types of coxsackieviruses, the method being for non-diagnostic and non-therapeutic purposes, the method using any one of the above kits for simultaneously detecting 4 types of coxsackieviruses, comprising the steps of:
(1) extracting RNA of a sample to be detected;
(2) taking RNA of a sample to be detected as a template, adding each component in the kit for simultaneously detecting the 4 types of coxsackie viruses to perform multiplex fluorescence quantitative PCR amplification reaction, performing detection analysis according to reaction results, and judging that the 4 types of coxsackie viruses are negative if S-type amplification curves do not appear in each detection channel; if the detection channel has an S-type amplification curve and the Ct value is less than or equal to 38, the corresponding coxsackie virus type is judged to be positive.
Preferably, the reaction system (25ul total system, the same applies below) for the multiplex quantitative PCR amplification reaction in the detection method for simultaneously detecting 4 types of coxsackieviruses comprises: 5mM dNTPs, 5 mu L of PCR reaction liquid, 0.32 mu mol of a coxsackie virus A9 type amplification primer pair, 0.32 mu mol of a coxsackie virus A10 type amplification primer pair, 0.32 mu mol of a coxsackie virus B2 type amplification primer pair, 0.32 mu mol of a coxsackie virus B5 type amplification primer pair, 0.06 mu mol of a coxsackie virus A9 type detection probe, 0.06 mu mol of a coxsackie virus A10 type detection probe, 0.06 mu mol of a coxsackie virus B2 type detection probe, 0.06 mu mol of a coxsackie virus B5 type detection probe, 1 mu L of enzyme mixture liquid and 0.5 mu L-5 mu L of sample RNA to be detected.
Preferably, in the detection method for simultaneously detecting 4 types of coxsackieviruses, the reaction conditions of the multiplex fluorescent quantitative PCR amplification reaction are as follows: reverse transcription is carried out for 10min to 30min at the temperature of 40 ℃ to 50 ℃; pre-denaturation at 93-95 ℃ for 2-10 min; denaturation at 93-95 ℃ for 10-30 s, annealing at 55-60 ℃, extension and signal acquisition for 30-60 s, and circulating for 40-45 times.
The multiple fluorescent quantitative PCR amplification reaction is carried out in a multiple fluorescent quantitative PCR instrument. Further, the multiple fluorescence quantitative PCR instrument is an ABI series multiple fluorescence quantitative PCR instrument, a Bio-Rad series (ICycler/MJ Opticon 2) multiple fluorescence quantitative PCR instrument, an S tratagene MX series multiple fluorescence quantitative PCR instrument, a Roche Lightcycler multiple fluorescence quantitative PCR instrument, a Ccpheid smartcycler multiple fluorescence quantitative PCR instrument, a Corbett Rortor-Gene multiple fluorescence quantitative PCR instrument and a Hangzhou Bori series multiple fluorescence quantitative PCR instrument. It should be noted that the multiplex quantitative PCR instrument is not limited to the PCR instrument mentioned above, and other multiplex quantitative PCR instruments can be used in the present invention.
Compared with the prior art, the invention has the following beneficial effects:
1. the detection primer pairs designed aiming at the 4 types of Coxsackie viruses are matched with each other, so that the mutual interference among the primers can be avoided, four items of Coxsackie virus A9 type, Coxsackie virus A10 type, Coxsackie virus B2 type and Coxsackie virus B5 type can be simultaneously detected for one tube of specimen through multiple fluorescence quantitative PCR, the detection time is saved, only one operation is needed, and the pollution generation opportunity is reduced.
2. The detection method using the kit has high sensitivity, and the detection sensitivity of the four viruses reaches 10 copies/mL. Meanwhile, the detection method of the invention has good specificity and does not have cross reaction with other viruses, such as coxsackie virus A2, rubella virus and measles virus.
3. The kit has very good amplification repeatability, and the precision obtained by calculation is less than or equal to 3 percent.
Drawings
FIG. 1 is a graph showing the amplification curves of positive samples of different concentrations in example 2;
FIG. 2 is a graph showing the amplification curves of the FAM channel of Coxsackie virus A9 at different concentrations of the positive test substance in example 2;
FIG. 3 is a graph showing the amplification of VIC channels of coxsackievirus A10 types at different concentrations of positive samples in example 2;
FIG. 4 is a graph showing the amplification curves of the TEXAS RED channel of Coxsackie virus type B2 at different concentrations of positive test substances in example 2;
FIG. 5 is a graph showing the amplification curves of CY5 channels of Coxsackie virus B5 at different concentrations of positive samples in example 2;
FIG. 6 is a graph showing the amplification curve of positive test samples in the experimental group of example 3;
FIG. 7 is a graph showing the amplification curve of the positive test sample of control group 1 in example 3;
FIG. 8 is a graph showing the amplification curve of the positive test sample of control group 2 in example 3;
FIG. 9 is a graph showing the amplification curve of the reproducibility test of the precision assay sample in example 4;
FIG. 10 is a graph showing the amplification curve of the FAM channel in the repetitive detection of coxsackievirus A9 type in samples to be tested with high precision in example 4;
FIG. 11 is a graph showing the amplification curve of the VIC channel in the repetitive detection of coxsackievirus A10 type in samples to be tested with precision in example 4;
FIG. 12 is a graph showing the amplification curve of the TEXAS RED channel in the repetitive detection of coxsackievirus B2 type in the samples to be tested with high precision in example 4;
FIG. 13 is a graph showing the amplification curve of CY5 channel in the repetitive assay of coxsackievirus B5 type in samples with high precision in example 4;
FIG. 14 is a graph showing the amplification curve of the positive test substance in example 5;
FIG. 15 is a graph showing the amplification curve of negative test substances in example 5;
FIG. 16 is a graph showing the amplification curves of the samples to be tested in example 5.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
The experimental procedures, in which specific conditions are not indicated in the examples, are usually carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer of the kits. The reagents used in the examples are all commercially available.
Example 1A kit for simultaneously detecting four types of Coxsackie viruses
The kit comprises RT-PCR reaction liquid, a nucleic acid composition for simultaneously detecting the four types of coxsackieviruses, enzyme mixed liquid, positive control and negative control. Wherein the RT-PCR reaction solution comprises 250mM Tris-base, 0.25% TritonX-100 by mass percent and 25mmol/L MgCl2。
The nucleic acid composition comprises:
a coxsackievirus A9 type amplification primer pair with the sequence shown as SEQ ID No.1 and SEQ ID No. 2;
a coxsackievirus A10 type amplification primer pair with the sequence shown as SEQ ID No.4 and SEQ ID No. 5;
a coxsackievirus B2 type amplification primer pair with the sequence shown as SEQ ID No.7 and SEQ ID No. 8;
a coxsackievirus B5 type amplification primer pair with the sequence shown as SEQ ID No.10 and SEQ ID No. 11;
the sequence of the detection probe is shown as SEQ ID No.3, wherein both ends of the detection probe of the coxsackievirus A9 type are respectively connected with FAM and BHQ1; the sequence of the detection probe is shown as SEQ ID No.6, the two ends of the detection probe of the coxsackie virus A10 type are respectively connected with VIC and BHQ1; a coxsackie virus B2 type detection probe with a sequence shown as SEQ ID No.9, wherein two ends of the coxsackie virus B2 type detection probe are respectively connected with TEXAS RED and BHQ2; the sequence of the detection probe is shown as SEQ ID No.12, the two ends of the detection probe of the coxsackievirus B5 type are respectively connected with CY5 and BHQ 2. The enzyme mixture comprises hot start Taq enzyme, MMLV reverse transcriptase and RNase inhibitor.
The positive control comprises RNA formed by reverse transcription of a plasmid containing a coxsackievirus A9 type target sequence, RNA formed by reverse transcription of a plasmid containing a coxsackievirus A10 type target sequence, RNA formed by reverse transcription of a plasmid containing a coxsackievirus B2 type target sequence and RNA formed by reverse transcription of a plasmid containing a coxsackievirus B5 type target sequence.
Specifically, the preparation process of the positive control is as follows: respectively taking the CA9 synthetic fragment, the CA10 synthetic fragment, the CB2 synthetic fragment and the B5 Coxsackie synthetic fragment as templates, and respectively amplifying corresponding amplified fragments by using corresponding primers; connecting the amplified products into a pUC57-Kan vector to respectively obtain a plasmid containing a coxsackie virus A9 type amplified fragment, a plasmid containing a coxsackie virus A10 type amplified fragment, a plasmid containing a coxsackie virus B2 type amplified fragment and a plasmid containing a coxsackie virus B5 type amplified fragment; and respectively carrying out reverse transcription on the plasmids to obtain corresponding RNA, namely a coxsackievirus A9 type positive standard substance, a coxsackievirus A10 type positive standard substance, a coxsackievirus B2 type positive standard substance and a coxsackievirus B5 type positive standard substance.
The negative control was a physiological saline solution containing none of coxsackievirus type a9, coxsackievirus type a10, coxsackievirus type B2, and coxsackievirus type B5.
Example 2 sensitivity of the kit for simultaneously detecting four types of Coxsackie viruses of example 1 was measured
(1) The coxsackievirus A9 type positive standard substance, the coxsackievirus A10 type positive standard substance, the coxsackievirus B2 type positive standard substance and the coxsackievirus B5 type positive standard substance in the kit of the embodiment 1 are mixed in equal amount to prepare a mixed solution, and positive samples to be tested are obtained (the concentration of each virus positive standard substance is 10)6copies/mL)。
(2) Carrying out 10-fold gradient dilution on the positive sample (namely 10 copies/mL-10)5copies/mL) was performed on the positive samples under each gradient using the kit of example 1, and the system of the multiplex quantitative PCR reaction is shown in Table 1. Wherein the volume ratio of the hot start Taq enzyme, the MMLV reverse transcriptase and the RNase inhibitor in the enzyme mixed solution is 4:1: 3. All reagents in the reaction system are placed in the same reaction tube for multiple fluorescent quantitative PCR detection. The reaction conditions of the multiplex fluorescent quantitative PCR reaction are as follows: reverse transcription at 50 deg.C for 15 min; pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 55 ℃, extension, signal acquisition for 40s, and circulation for 40 times.
TABLE 1 System for multiplex fluorescent quantitative PCR reactions
| Reagent | Concentration of | Volume (mu L/portion) |
| dNTPs | 2.5mM/ |
2 |
| PCR reaction solution | -- | 5 |
| Upstream primer of A9 type coxsackie virus amplification primer pair | 50pmoL/μL | 0.16 |
| Downstream primer of A9 type coxsackie virus amplification primer pair | 50pmoL/μL | 0.16 |
| Upstream primer of A10 type coxsackie virus amplification primer pair | 50pmoL/μL | 0.16 |
| Downstream primer of A10 type coxsackie virus amplification primer pair | 50pmoL/μL | 0.16 |
| Upstream primer of B2 type coxsackie virus amplification primer pair | 50pmoL/μL | 0.16 |
| Downstream primer of B2 type coxsackie virus amplification primer pair | 50pmoL/μL | 0.16 |
| Upstream primer of B5 type coxsackie virus amplification primer pair | 50pmoL/μL | 0.16 |
| Downstream primer of B5 type coxsackie virus amplification primer pair | 50pmoL/μL | 0.16 |
| A9 type coxsackie virus detection probe | 50pmoL/μL | 0.06 |
| A10 type coxsackie virus detection probe | 50pmoL/μL | 0.06 |
| B5 type coxsackie virus detection probe | 50pmoL/μL | 0.06 |
| B2 type coxsackie virus detection probe | 50pmoL/μL | 0.06 |
| Enzyme mixture | -- | 1 |
| RNA of sample to be tested | -- | 5 |
| ddH2O | -- | To a total volume of 25 |
(3) And (4) analyzing results: amplification of a curve by fluorescenceAnd judging whether the detection result is positive or negative according to the line graph and the Ct value, and determining whether the sample contains coxsackie virus A9, coxsackie virus A10, coxsackie virus B2 and coxsackie virus B5. Specifically, when the Ct value of the amplification curve graph is less than or equal to 38 and the amplification curve shows obvious exponential increase (i.e. S type), the result is positive; when Ct value of amplification curve graph>38 or no Ct value, the result was negative. The detection results are shown in FIGS. 1 to 5. FIG. 1 is a graph showing the amplification curve of positive samples of different concentrations in example 2. FIG. 2 is a graph showing the amplification curves of the FAM channel of Coxsackie virus A9 type at different concentrations of positive samples in example 2, and the curves indicated by the arrows (2-1), (2-2), (2-3), (2-4) and (2-5) in FIG. 2 correspond to concentrations of 105copies/m L、104copies/mL、103copies/mL、102Amplification curves at copies/mL, 10 copies/mL. FIG. 3 is a graph showing the amplification curves of the VIC channel of coxsackievirus A10 type of positive samples with different concentrations in example 2, and the curves indicated by the arrow (3-1), the arrow (3-2), the arrow (3-3), the arrow (3-4) and the arrow (3-5) in FIG. 3 correspond to concentrations of 10 respectively5copies/mL、104copies/mL、103copies/mL、102Amplification curves at copies/mL, 10 copies/mL. FIG. 4 is a graph showing the amplification curves of the Texas RED channel of Coxsackie virus type B2 at different concentrations of positive samples in example 2, and the curves indicated by the arrows (4-1), (4-2), (4-3), (4-4) and (4-5) in FIG. 4 correspond to concentrations of 105copies/mL、104copies/mL、103copies/mL、102Amplification curves at copies/mL, 10 copies/mL. FIG. 5 is a graph showing the amplification curves of CY5 channel of coxsackievirus type B5 with different concentrations of positive samples in example 2, and the curves indicated by arrow (5-1), arrow (5-2), arrow (5-3), arrow (5-4) and arrow (5-5) in FIG. 5 correspond to concentrations of 105copies/mL、104copies/mL、103copies/mL、102Amplification curves at copies/mL, 10 copies/mL.
As can be seen from FIG. 1, the kit of example 1 can simultaneously detect Coxsackie virus A9 and Coxsackie virus in a sample to be detected at one timeThe detection time is saved by the A10 type, the Coxsackie virus B2 type and the Coxsackie virus B5 type. As can be seen from FIGS. 2 to 5, the coxsackie virus A9, coxsackie virus A10, coxsackie virus B2 and coxsackie virus B5 are 10 copies/mL-105The amplification curves in the range of copies/mL all present good linear relation, the detection sensitivity of the four types of the coxsackieviruses all reaches 10copies/mL, and the sensitivity is higher.
[ example 3 ]
(1) The kit of example 1 was used to prepare a coxsackievirus A9 type positive standard, a coxsackievirus A10 type positive standard, a coxsackievirus B2 type positive standard, and a coxsackievirus B5 type positive standard (each virus positive standard was used at a concentration of 10%6copies/mL) and preparing a mixed solution to obtain a positive to-be-detected product; the normal saline without the positive reference substance of the four types of coxsackie viruses is a negative sample to be tested.
(2) The experiment was divided into an experimental group, a control group 1 and a control group 2, and the kit of the experimental group was the kit of example 1. The kit components of control 1 and control 2 were commercial kits in use, and comprised: dNTPs, PCR reaction liquid, amplification primers and probes of four viruses, enzyme mixed liquid, positive standard substances and negative standard substances. The kits of the control groups 1 and 2 were substantially the same as the kit of example 1.
(3) The kit of the experimental group, the kit of the control group 1 and the kit of the control group 2 are adopted to carry out multiple fluorescence quantitative PCR detection on the positive to-be-detected product and the negative to-be-detected product, and the reaction system, the reaction conditions and the result analysis are the same as those in the example 2. The detection results are shown in FIGS. 6 to 8. Wherein, fig. 6 is an amplification curve chart of the positive test sample of the experimental group in example 3, a curve indicated by an arrow (6-1) in fig. 6 is an amplification curve of coxsackievirus type a9, a curve indicated by an arrow (6-2) in fig. 6 is an amplification curve of coxsackievirus type a10, a curve indicated by an arrow (6-3) in fig. 6 is an amplification curve of coxsackievirus type B2, and a curve indicated by an arrow (6-4) in fig. 6 is an amplification curve of coxsackievirus type B5. FIG. 7 is a graph showing an amplification curve of a positive test sample of the control group 1 in example 3, in which the curve indicated by the arrow (7-1) in FIG. 7 is an amplification curve of coxsackie virus type A9, the curve indicated by the arrow (7-2) in FIG. 7 is an amplification curve of coxsackie virus type A10, the curve indicated by the arrow (7-3) in FIG. 7 is an amplification curve of coxsackie virus type B2, and the curve indicated by the arrow (7-4) in FIG. 7 is an amplification curve of coxsackie virus type B5. FIG. 8 is a graph showing the amplification curve of the positive test sample of the control group 2 in example 3, in which the curve indicated by the arrow (8-1) in FIG. 8 is an amplification curve of Coxsackie virus type A9, the curve indicated by the arrow (8-2) in FIG. 8 is an amplification curve of Coxsackie virus type A10, and the curve indicated by the arrow (8-4) in FIG. 8 is an amplification curve of Coxsackie virus type B5.
As can be seen from fig. 6, the experimental group obtains four S-type amplification curves, and the negative test sample has no amplification curve, which indicates that the kit of the experimental group can simultaneously detect coxsackie virus a9, coxsackie virus a10, coxsackie virus B2 and coxsackie virus B5 in the positive test sample, and simultaneously, no non-specific amplification curve occurs.
As can be seen from fig. 7, the control group 1 obtains four S-type amplification curves, which indicates that the control group 1 can simultaneously detect coxsackie virus a9, coxsackie virus a10, coxsackie virus B2 and coxsackie virus B5 in the sample to be detected, but a false positive amplification curve exists, which indicates that false positives easily occur when four pairs of primers and four probes of the control group 1 are subjected to virus detection.
As can be seen from FIG. 8, three S-shaped amplification curves were obtained in control group 2, and the positive standard of Coxsackie virus B2 was not amplified, indicating that the four pairs of primers and four probes in control group 1 interfere with each other, resulting in failure to detect the four types of Coxsackie viruses simultaneously.
Example 4 precision of the kit for simultaneously detecting four types of Coxsackie virus of example 1 was measured
The positive standard substance of coxsackie virus A9, the positive standard substance of coxsackie virus A10, the positive standard substance of coxsackie virus B2 and the positive standard substance of coxsackie virus B5 in the kit of the embodiment 1 are mixed and prepared into mixed liquid, and the positive to-be-detected product is obtained. Carrying out 10-fold gradient dilution on the positive sample (the concentration gradient is 10)5copies/mL、104copies/mL、103copies/mL、102copies/mL, 10copies/mL) to obtain the precision sample.
The kit used in example 1 was used for multiplex fluorescence quantitative PCR amplification with 10 duplicate wells of the sample to be tested for amplification precision, and the results are shown in FIGS. 9-13. FIG. 9 is a graph showing the amplification curve of the reproducibility test of the precision assay sample in example 4. FIG. 10 is a graph showing the amplification curve of the FAM channel in the repetitive detection of coxsackievirus A9 type in samples with high precision in example 4. FIG. 11 is a graph showing the amplification of VIC channel in the repeated detection of coxsackievirus A10 in the samples with high precision in example 4. FIG. 12 is a graph showing the amplification of the TEXAS RED channel in repeated detection of coxsackievirus B2 type as a precision test substance in example 4. FIG. 13 is a graph showing the amplification curve of CY5 channel in the repetitive detection of coxsackievirus B5 type in samples with high precision in example 4.
As can be seen from FIGS. 9 to 13, the precision of each channel of the kit of the above embodiment is very good, the CT value data is derived, the precision obtained by calculation is less than or equal to 3%, and the amplification repeatability of the kit is very good.
Example 5 the specificity of the kit for simultaneously detecting coxsackie virus type IV of example 1 was determined
(1) Mixing the coxsackievirus A9 type positive standard substance, the coxsackievirus A10 type positive standard substance, the coxsackievirus B2 type positive standard substance and the coxsackievirus B5 type positive standard substance in the kit of the embodiment 1 to prepare a mixed solution to obtain a positive to-be-detected substance, wherein the concentration of the positive to-be-detected substance is 105copies/mL. The negative control was physiological saline without coxsackievirus A9 type gene, coxsackievirus A10 type gene, coxsackievirus B2 type gene and coxsackievirus B5 type gene.
(2) The samples to be extracted are 15 samples which are 3 samples of coxsackievirus A9, 1 sample of coxsackievirus A10, 3 samples of coxsackievirus B2, 4 samples of coxsackievirus B5, 1 sample of coxsackievirus A2, 2 samples of rubella virus and 1 sample of measles virus, and all the samples are from a disease control center. And respectively extracting RNA in each sample to be extracted by adopting a virus RNA extraction kit (purchased from Tiangen Biochemical technology (Beijing) Co., Ltd.) to obtain corresponding samples to be detected.
(3) And (3) carrying out multiple fluorescent quantitative PCR detection on the positive test substance, the negative control test substance and each test sample in the step (2) by using the kit in the embodiment 1, wherein the reaction system, the reaction conditions and the result analysis are the same as those in the embodiment 2. The measurement results are shown in FIGS. 14 to 16. FIG. 14 is a graph showing an amplification curve of a positive test substance in example 5, in which the curve indicated by the arrow (14-1) in FIG. 14 is an amplification curve of Coxsackie virus type A9, the curve indicated by the arrow (14-2) in FIG. 14 is an amplification curve of Coxsackie virus type A10, the curve indicated by the arrow (14-2) in FIG. 14 is an amplification curve of Coxsackie virus type B2, and the curve indicated by the arrow (14-4) in FIG. 14 is an amplification curve of Coxsackie virus type B5. FIG. 15 is a graph showing the amplification curve of the negative control test substance in example 5. FIG. 16 is a graph showing the amplification curves of the samples to be tested in example 5.
As can be seen from FIGS. 14 to 15, the kit of example 1 can detect four viruses in the positive test sample, and the negative test sample is not detected, indicating that the kit of example 1 has high specificity.
As can be seen from fig. 16, when the kit of example 1 is used to detect a sample to be detected, 3 samples of coxsackie virus type a9, 1 sample of coxsackie virus type a10, 3 samples of coxsackie virus type B2 and 4 samples of coxsackie virus type B5 can be detected; the coxsackievirus A2, rubella virus and measles virus are not detected, and the accuracy rate of the detection sample reaches 100%.
In summary, the kit for simultaneously detecting four types of coxsackieviruses in the above embodiment can simultaneously detect four items of coxsackievirus A9, coxsackievirus A10, coxsackievirus B2 and coxsackievirus B5 for one tube of specimen, has the advantages of short detection time, high detection sensitivity, high accuracy, good specificity and good repeatability, and has higher guiding significance for clinical application.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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Claims (10)
1.4 detection primers and probes for coxsackie virus nucleic acid of type 4, which are characterized by comprising specific amplification primer sequences of type 4 genes and specific fluorescent probe sequences corresponding to the primers, wherein the 5 'end of the probe sequences is marked with a fluorescent reporter group, the 3' end of the probe sequences is marked with a fluorescent quenching group, and the detection primers and the probe sequences of the types are as follows:
coxsackievirus type a 9:
an upstream primer CVA 9-F: the nucleotide sequence is shown as SEQ ID NO.1,
the nucleotide sequence of the downstream primer CVA9-R is shown as SEQ ID NO 2,
the nucleotide sequence of the specific probe CVA9-P is shown as SEQ ID NO 3,
coxsackievirus type a 10:
an upstream primer CVA 10-F: the nucleotide sequence is shown as SEQ ID NO.4,
the nucleotide sequence of the downstream primer CVA10-R is shown as SEQ ID NO.5,
the nucleotide sequence of the specific probe CVA10-P is shown as SEQ ID NO 6,
coxsackievirus B2 type:
an upstream primer CVB 2-F: the nucleotide sequence is shown as SEQ ID NO.7,
the nucleotide sequence of the downstream primer CVB2-R is shown as SEQ ID NO 8,
the nucleotide sequence of the specific probe CVB2-P is shown as SEQ ID NO 9,
coxsackievirus B5 type:
an upstream primer CVB 5-F: the nucleotide sequence is shown as SEQ ID NO.10,
the nucleotide sequence of the downstream primer CVB5-R is shown as SEQ ID NO.11,
the nucleotide sequence of the specific probe CVB5-P is shown as SEQ ID NO. 12.
2. The detection primers and probes for 4 types of coxsackie virus nucleic acids according to claim 1, wherein the fluorescent reporter group carried by the coxsackie virus A9 type detection probe, the coxsackie virus A10 type detection probe, the coxsackie virus B2 type detection probe and the coxsackie virus B5 type detection probe is selected from at least one of 6-carboxyfluorescein, hexachloro-6-methylfluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxy-X-rhodamine, 6-carboxytetramethylrhodamine, sulforhodamine, 6-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein succinimidyl ester, cyanine3, cyanine3.5, cyanine5 and cyanine 5.5; the fluorescence quenching group carried by the detection probe of the coxsackievirus A9 type, the detection probe of the coxsackievirus A10 type, the detection probe of the coxsackievirus B2 type and the detection probe of the coxsackievirus B5 type is at least one selected from 6-carboxytetramethylrhodamine, 4- (4-dimethylamino phenylazo) benzoic acid, a black hole quenching agent 1, a black hole quenching agent 2 or a black hole quenching agent 3.
3. The primers and probes for detecting the type 4 coxsackie virus nucleic acid as claimed in claim 2, wherein the fluorescent reporter group carried by the coxsackie virus A9 type detection probe, the coxsackie virus A10 type detection probe, the coxsackie virus B2 type detection probe and the coxsackie virus B5 type detection probe is selected from one of 6-carboxyfluorescein, VIC fluorescent dye, sulforhodamine and sulforhodamine; the fluorescence quenching group is selected from one of a black hole quenching agent 1 or a black hole quenching agent 2; preferably, the fluorescence reporter group of the Coxsackie virus A9 type detection probe is 6-carboxyfluorescein, the fluorescence quenching group is BHQ1, the fluorescence reporter group of the Coxsackie virus A10 type detection probe is VIC fluorescent dye, the fluorescence quenching group is BHQ1, the fluorescence reporter group of the Coxsackie virus B2 type detection probe is sulforhodamine, the fluorescence quenching group is BHQ2, the fluorescence reporter group of the Coxsackie virus B5 type detection probe is sulforhodamine, and the fluorescence quenching group is BHQ 2.
4. A kit for simultaneously detecting 4 types of coxsackieviruses, which is characterized by comprising detection primers and probes of the 4 types of coxsackieviruses of any one of claims 1 to 3, wherein the detection probes of the coxsackievirus A9, the coxsackievirus A10, the coxsackievirus B2 and the coxsackievirus B5 are respectively provided with different fluorescent reporter groups.
5. The kit for simultaneously detecting 4 types of coxsackie viruses according to claim 4, wherein when in use, the kit is prepared by mixing the following primers and probes according to the following proportion: the molar ratio of the amplification primer pair of the coxsackievirus A9 to the detection probe of the coxsackievirus A9 is 5:1, the molar ratio of the amplification primer pair of the coxsackievirus A10 to the detection probe of the coxsackievirus A10 is 5:2, the molar ratio of the amplification primer pair of the coxsackievirus B2 to the detection probe of the coxsackievirus B2 is 2:1, the molar ratio of the amplification primer pair of the coxsackievirus B5 to the detection probe of the coxsackievirus B5 is 10:3, and the molar ratio of the detection probe of the coxsackievirus A9, the detection probe of the coxsackievirus A10, the detection probe of the coxsackievirus B2 and the detection probe of the coxsackievirus B5 is 2:4:5: 6.
6. The kit for simultaneously detecting coxsackie viruses of 4 types according to claim 4, wherein the kit further comprises a positive quality control product and a negative quality control product, wherein the negative quality control product is a physiological saline solution, and the positive control product comprises RNA formed by reverse transcription of a plasmid containing a coxsackie virus A9 type target sequence, RNA formed by reverse transcription of a plasmid containing a coxsackie virus A10 type target sequence, RNA formed by reverse transcription of a plasmid containing a coxsackie virus B2 type target sequence and RNA formed by reverse transcription of a plasmid containing a coxsackie virus B5 type target sequence.
7. The kit for simultaneously detecting 4 types of Coxsackie viruses according to claim 4, wherein the kit further comprises at least one of RNA extraction reagents, dNTPs, PCR reaction solution and enzyme mixed solution, wherein the PCR reaction solution comprises 220 mM-280 mM Tris-base, 0.2-0.3% TritonX-100 by mass and 20-30 mmol/L MgCl2(ii) a The enzyme mixed solution comprises an RNA enzyme inhibitor, Taq enzyme and reverse transcriptase.
8. The kit for simultaneously detecting 4 types of Coxsackie viruses according to claim 7, wherein the PCR reaction solution of the kit comprises 250mM Tris-base, 0.25% TritonX-100 by mass and 25mmol/L MgCl2(ii) a The Taq enzyme in the enzyme mixed solution is hot start Taq enzyme, the reverse transcriptase is MMLV reverse transcriptase, and the volume ratio of the RNase inhibitor to the hot start Taq enzyme to the reverse transcriptase is 3:4: 1.
9. Use of the kit for simultaneous detection of 4 type coxsackie viruses according to any one of claims 4-8 in the preparation of a reagent for detecting 4 type coxsackie viruses.
10. A detection method for simultaneously detecting 4 types of Coxsackie viruses, which is used for non-diagnostic and non-therapeutic purposes, comprising the following steps:
(1) extracting RNA of a sample to be detected;
(2) taking RNA of a sample to be detected as a template, adding each component in the kit for simultaneously detecting the 4 types of coxsackie viruses according to any one of claims 4-8 to perform multiple fluorescent quantitative PCR amplification reaction, performing detection analysis according to the reaction result, and judging that the 4 types of coxsackie viruses are negative if an S-shaped amplification curve does not appear in a detection channel; if the detection channel has an S-type amplification curve and the Ct value is less than or equal to 38, determining the corresponding coxsackie virus type as positive;
the reaction conditions of the multiple fluorescent quantitative PCR amplification reaction in the detection method for simultaneously detecting the 4 types of Coxsackie viruses are as follows: reverse transcription is carried out for 10min to 30min at the temperature of 40 ℃ to 50 ℃; pre-denaturation at 93-95 ℃ for 2-10 min; denaturation at 93-95 ℃ for 10-30 s, annealing at 55-60 ℃, extension and signal acquisition for 30-60 s, and circulating for 40-45 times.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748650A (en) * | 2020-06-23 | 2020-10-09 | 深圳市艾伟迪生物科技有限公司 | Nucleic acid composition for simultaneously detecting four B-group coxsackie virus subtypes and kit and detection method thereof |
| CN112593011A (en) * | 2020-12-25 | 2021-04-02 | 中山大学 | Primer and probe for detecting coxsackie virus B group |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101500591A (en) * | 2005-09-08 | 2009-08-05 | Mnd诊断有限公司 | Compositions and methods using same for the detection of viruses |
| CN101886138A (en) * | 2009-05-11 | 2010-11-17 | 北京索奥生物技术有限公司 | Three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as kit thereof |
| CN101892324A (en) * | 2010-07-05 | 2010-11-24 | 中国人民武装警察部队医学院 | Primer set for use in detection of enteroviruses by loop-mediated isothermal amplification technique and detection system |
| CN107513584A (en) * | 2017-09-25 | 2017-12-26 | 浙江大学 | A kind of five heavy fluorescence quantitative kits for detecting enterovirus |
| CN109504805A (en) * | 2018-12-28 | 2019-03-22 | 深圳市艾伟迪生物科技有限公司 | The nucleic acid compositions and kit and detection method of four kinds of enteroviruses of detection simultaneously |
| CN110184242A (en) * | 2019-06-11 | 2019-08-30 | 武汉生物制品研究所有限责任公司 | The mouse strong virus force of 6 type of Coxsackie virus A group (CVA6) attacks strain and its application |
-
2019
- 2019-10-21 CN CN201911002544.0A patent/CN110863065A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101500591A (en) * | 2005-09-08 | 2009-08-05 | Mnd诊断有限公司 | Compositions and methods using same for the detection of viruses |
| CN101886138A (en) * | 2009-05-11 | 2010-11-17 | 北京索奥生物技术有限公司 | Three-color fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) combined detection method of enterovirus 71, Coxsackie virus A16 and other subtypes of enterovirus as well as kit thereof |
| CN101892324A (en) * | 2010-07-05 | 2010-11-24 | 中国人民武装警察部队医学院 | Primer set for use in detection of enteroviruses by loop-mediated isothermal amplification technique and detection system |
| CN107513584A (en) * | 2017-09-25 | 2017-12-26 | 浙江大学 | A kind of five heavy fluorescence quantitative kits for detecting enterovirus |
| CN109504805A (en) * | 2018-12-28 | 2019-03-22 | 深圳市艾伟迪生物科技有限公司 | The nucleic acid compositions and kit and detection method of four kinds of enteroviruses of detection simultaneously |
| CN110184242A (en) * | 2019-06-11 | 2019-08-30 | 武汉生物制品研究所有限责任公司 | The mouse strong virus force of 6 type of Coxsackie virus A group (CVA6) attacks strain and its application |
Non-Patent Citations (1)
| Title |
|---|
| 左安琳;: "小儿手足口病312例临床分析" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748650A (en) * | 2020-06-23 | 2020-10-09 | 深圳市艾伟迪生物科技有限公司 | Nucleic acid composition for simultaneously detecting four B-group coxsackie virus subtypes and kit and detection method thereof |
| CN112593011A (en) * | 2020-12-25 | 2021-04-02 | 中山大学 | Primer and probe for detecting coxsackie virus B group |
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Application publication date: 20200306 |