CN108486259A - One-step method detects the kit and detection method of pertussis nucleic acid - Google Patents

One-step method detects the kit and detection method of pertussis nucleic acid Download PDF

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CN108486259A
CN108486259A CN201810172491.6A CN201810172491A CN108486259A CN 108486259 A CN108486259 A CN 108486259A CN 201810172491 A CN201810172491 A CN 201810172491A CN 108486259 A CN108486259 A CN 108486259A
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sample
nucleic acid
magnetic bead
kit
pcr
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胡彬
刘杰
蔡媛媛
陶施芳
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Shaoxing Xun Xun Kang Biological Technology Co Ltd
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Shaoxing Xun Xun Kang Biological Technology Co Ltd
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Abstract

The invention discloses a kind of kit for detecting nucleic acid of Bordetella pertussis in detection sample, the kit includes nucleic acid cleavage solution, wherein, nucleic acid cleavage solution includes magnetic bead, the nucleic acid cleavage solution includes 1M NaCl, 0.01%Triton X and 0.001M KCl, the hydroxyl magnetic bead that magnetic bead is 0.005 milligram.It is that kit can be with one-step method extraction nucleic acid and without additional step using this, full-automatic operation may be implemented in the augmentation detection of progress nucleic acid that can be quick and easy.

Description

One-step method detects the kit and detection method of pertussis nucleic acid
Technical field
The invention belongs to molecular diagnostics biological technical fields, are related to one-step method nucleic acid extraction and fluorescence quantitative PCR detection Kit, and in particular to the real-time fluorescence quantitative PCR reagent containing probe detects patient's nasopharynx reagent, one hundred days in sputum equal samples The kit for detecting nucleic acid of cough bacillus.
Background technology
Pertussis is a kind of mainly Acute Respiratory communicable disease caused by Bordetella pertussis.Bordetella pertussis (Bordetella pertussis) is a kind of Gram-negative dialister bacterium, belongs to special Salmonella.The bacterium is mainly passed by air It broadcasts, there is highly infectious and artificial unique host.Pertussis is apt to occur in infant and age small children, and clinical manifestation is Clonic spasm is coughed, and vomiting, the course of disease typically lasts for several weeks, the easy Complicating Pneumonia In Patients in baby channel, epilepsy, so as to cause death.Often There are 20,000,000-4,000 ten thousand pertussis cases in year whole world, wherein the death more than 350,000, and mainly infant.
Although currently, there is pertussis vaccine, the disease detection of WTO finds the pertussis brought after long-term vaccine inoculation The incidence trend of declining to a great extent reverses, and outstanding behaviours is that developed country the multiple liter of incidence and developing country occurs repeatedly Pertussis outburst occurs.Pertussis need to be differentiated with respiratory disease, such as bronchitis, pneumonia etc.；Especially feel in adult The clinical manifestation for contaminating pertussis is very easy to mistaken diagnosis as bronchitis, so as to cause mistaken diagnosis, infects household.Therefore pertussis diagnosis is non- It is often important
The method of existing traditional scheme detection Bordetella pertussis mainly has following：1. pertussis culture is pertussis detection Goldstandard, high specificity, but its culture period length (generally about 7d), sensitivity it is low and to culture technique height, be not suitable for The quick detection in laboratory.The immunological detection method of pertussis often takes 2 parts of serum specimens of suspected acute phase and convalescence to carry out Detection, the sampling period is long, and sensitivity and specificity are not high, is not suitable for the quick diagnosis of pertussis.Pertussis it is fast Speed is diagnosed as PCR diagnosis, and PCR diagnosis are extracted firstly the need of to sample nucleic acid, and the extracting mode of mainstream carries out for paramagnetic particle method Extraction.
But magnetic bead extracts nucleic acid, traditional extracting method step is complicated, and time-consuming, it is not easy to realize automation Detection.
Invention content
In view of the above-mentioned problems, since Bordetella pertussis value-added speed is fast, lesion can be caused.So we providing a kind of magnetic Pearl method cracks the one-step method kit of absorption to the progress Rapid nucleic acid in sample and keeps preferable detection sensitivity.Utilize magnetic Pearl directly extracts sample, without being washed to magnetic bead, directly carries out nucleic acid with magnetic bead and the contact of the reagent of amplification Amplification, reduces operating procedure, meanwhile, avoid interference of the impurity to detection.In addition, sample need not also do excessive place Reason, simplifies mode, and the platform that automation may be implemented is operated.
One aspect of the present invention provides the kit for detecting nucleic acid of a detection Bordetella pertussis, which includes lysate And bead suspension, wherein the ingredient of lysate includes 1M NaCl, 0.01%Triton-X and 0.001M KCl, and magnetic bead suspends Liquid includes the magnetic bead of hydroxyl modified, polydispersity coefficient ＜ 0.2；Magnetic bead is that diameter is less than or equal to 500nM, wherein magnetic bead contains Amount is 10mg/mL.
In some preferred modes, when lysate and the mixing of magnetic bead solution, the volume ratio of the two is 200:1.
Alternatively, providing a kind of mixture, which is solution mixture, which includes 1M NaCl, 0.01% The hydroxyl magnetic bead of Triton-X and 0.001M KCl and 0.005 milligram of 10mg/mL.
Alternatively, providing magnetic bead lysate in the way of table 1, the magnetic bead lysate is by lysate and magnetic bead liquid according to volume 200：1 mode mixes.
In some preferred modes, which further includes the necessary ingredient of PCR reaction amplifications, the amplification Reagent includes the Mg2+ of 1~10mmol/L, the Tris-HCl buffer solutions of 10~50mmol/L；The DNTP of 50~1000umol/L； The K ions of concentration 25mmol/L, the enzyme stability reagent of 100 μ g/ml, such as BSA etc..Preferably, it is also wrapped in amplifing reagent Include glycerine.Further include special probe sequence in some preferred modes, the sequence be SEQ.1.1-1.3 and SEQ.2.1-2.3。
On the other hand, the object of the present invention is to provide a kind of one-step method nucleic acid extractions and the detection of nucleic acids of Bordetella pertussis to try Agent box, wherein including magnetic bead lysate, quantitative PCR detection liquid to kit.Preferably, which further includes：Negative control, Positive control, specification and box body composition.Specific component see the table below.
Table 1：The component and volume ratio of magnetic bead lysate.
Table 2:The composition of amplifing reagent and specific ingredient
Preferably, quantitative PCR detection liquid contains PCR buffer solutions, hot resistant DNA polymerase, three kinds of primer and probes.Described Primer is specifically, being table 3.
Table 3：Fluorescent quantitative PCR primed probe
In some modes, further include negative control be TE buffer；Positive control is synthesis Bordetella pertussis target sequence Plasmid sample.Kit of the present invention should be stored in -20 DEG C (wherein magnetic bead lysate 2-8 DEG C placement), multigelation 8 times with It is interior.
On the one hand, the present invention provides a kind of method of the detection of nucleic acids of detection Bordetella pertussis, and this method includes providing The reagent of table 1-3, the extraction step of nucleic acid and the amplification step of nucleic acid, wherein the extraction of nucleic acid includes：One, nucleic acid DNA carries It takes：
1. magnetic bead lysate is taken out, oscillation shakes up rear of short duration centrifugation；
2. taking the internal control of magnetic bead lysate and the μ of n × 1 L that the μ of n × 80 L are added in suitable centrifuge tube or PCR pipe, mixing； 3. in the centrifuge tube of step 2 or PCR pipe, often 20 μ L samples (sample size of N expression processing or the number of PCR pipe is added in pipe Amount)；
4. test should at least be arranged a hole negative control and a hole positive control every time, the same sample of loading methods, it is negative and The method of positive method for extracting nucleic acid and detection sample is consistent.
5. covering pipe lid, centrifuge tube or PCR pipe are placed in 80 DEG C of constant temperature and are incubated 5min by of short duration centrifugation, and then room temperature is put Set 5min.If so, eight unions can be put into PCR instrument, 80 DEG C × 5min is set, 20 DEG C × 5min.6. by step 5 centrifuge tube or Eight unions, which are placed on magnetic frame, stands 1-2min；Liquid is removed, magnetic bead is retained, while magnetic bead is not done any further clear It washes, dissolves, washs or handle.If being accidentally drawn onto magnetic bead, then liquid is returned in pipe, is inhaled again after standing 1min again.It inhales It is complete to abandon liquid, or take out liquid using automated process, retains magnetic bead in PCR pipe.It adsorbs on magnetic bead this when Have nucleic acid-templated, while other impurity may also be adsorbed with.
Two：Fluorescent quantitative PCR：
7. with liquid-transfering gun draw 50 μ L kits 2 in quantitative PCR detection liquid, be added separately to step 6 centrifuge tube or In eight unions.
(please note that part pipette tips may adsorb magnetic bead, it will influence 8. being inhaled repeatedly with liquid-transfering gun and beating to magnetic bead to be completely dispersed Testing result, mixing here can not also use liquid-transfering gun, but the method for using vibrations mixes automatically).Such as be 1.5mL from Heart pipe must be transferred in PCR pipe or PCR disks.Close the lid or seal up glued membrane.
9. being immediately placed in PCR instrument carries out augmentation detection.If do not detected temporarily, PCR pipe or PCR disks must be kept in dark place in 2- It is no more than 2 hours in 8 DEG C of refrigerators.
10. loop parameter below is arranged in PCR instrument：
95℃×10min；95 DEG C × 15s, 60 DEG C × 45s are pressed again to recycle 40 times；
Fluoroscopic examination is at 60 DEG C；Reaction system is 50 μ L
11. fluorescence channel detection selects and (collects fluorescence FAM and CY5)；If with the PCR instrument of ABI series, Quencher Dye selects None.11. save file runs program.
12. experiment terminates, suitable threshold line is set, obtains Ct values, judgement sample feminine gender positive findings.
Sample in all modes can be liquid type sample：Serum, urine, mouthwash, hydrothorax, ascites, cerebrospinal fluid, room Water is used directly for nucleic acid extraction；Or swab sample, such as Nasopharyngeal swabs, sputum class sample, swab or sputum sample It is dissolved in 1-5mL physiological saline.
Advantageous effect：
1. pair nucleic acid carries out easy rapid extraction：With magnetic bead adsorption of DNA nucleic acid, without washing elution, to be directly used in PCR anti- It answers；2. desired sample size is few：Nucleic acid extraction sample in general existing traditional technology is 1mL, and to sample in the present invention It is required that being 20ul or can less realize；3. being used for quickly detecting to Bordetella pertussis by quantitative fluorescent PCR, have very well Specificity and sensitivity.
Description of the drawings
Fig. 1 is the experimental result picture of the detection positive or negative sample in a specific implementation mode of the invention, wherein No. 1 curve is positive control, and No. 2 curves are positive sample (a concentration of 106CFU/ml), and 3-5 is respectively negative control and 2 the moon Property sample curve.
Fig. 2 is the signal graph of specific detection experiment, wherein No. 1 curve is positive control, and 2-7 curves are respectively the moon Property control, influenza A virus (National reference), influenza B virus (National reference), Escherichia coli (ATCC35150), Staphylococcus aureus (ATCC6538) and Candida albicans (ATCC10231) positive sample.
Fig. 3 is to positive sample sensitivity technique signal graph, wherein No. 1, No. 2, No. 3 and No. 4 curve is respectively ATCC 9340 bordetella pertussis concentration are respectively 106CFU/ml、105CFU/ml、104CFU/ml and 103CFU/ml samples.
Specific implementation mode
Below in conjunction with the drawings and specific embodiments, invention is further described in detail, and the scheme that embodiment provides is Preferred embodiment is how to be put into practice as those of ordinary skill in the art marrow according to the invention to crack, but not as right The restriction of the application, scope of the present application embody in the claims.
Explanation：The PCR amplification instrument used in following embodiment is Bio-RAD CFX96 and ABI 7500.
Embodiment 1：The sample of Bordetella pertussis of the magnetic bead one-step method detection by the confirmation positive or negative sample
The reagent of offer such as following table：
Table 1：The component and volume ratio of magnetic bead lysate.
Table 2:The composition of amplifing reagent and specific ingredient
Table 3：Fluorescent quantitative PCR primed probe
The specific method is as follows：
One, nucleic acid DNA extracts：
1. the magnetic bead lysate in such as table 1 is taken out, oscillation shakes up rear of short duration centrifugation.
2. taking suitable centrifuge tube that the magnetic bead lysate of 5 × 80 μ L, mixing is added.Then it uses liquid-transfering gun according to 80 μ L/ pipes, divides Be not added in eight unions of PCR in 5 holes, 1 be used for positive sample nucleic acid extraction, 2 be used for negative sample nucleic acid extraction, 2 It is a to be used for negative and positive control.
3. in the PCR pipe of step 2, it is separately added into 20ul or less samples：
5 experiment samples Sample type
Negative sample TE buffer
Positive control Synthesize target sequence plasmid
Positive sample ATCC9430(106CFU/ml) (commercially available acquisition)
Negative sample Clinical sample (is stored in the throat swab sample in physiological saline)
Positive sample Clinical sample (is stored in the throat swab sample in physiological saline)
4. covering pipe lid, eight unions are placed in 80 DEG C of constant temperature and are incubated 5min, are then placed at room temperature for 5min by of short duration centrifugation.
5. step 4 centrifuge tube or eight unions to be placed on magnetic frame and stand 1-2min.Liquid is carefully sucked with pipettor. If being accidentally drawn onto magnetic bead, then liquid is returned in pipe, inhaled again after standing 1min again, suction is abandoned liquid and only to be retained completely Magnetic bead in test tube, does not carry out magnetic bead the additional of any steps such as the nucleic acid of subsequent cleaning, washing, dissolving magnetic bead absorption Processing.
6. the above-mentioned PCR of 50ul, which are added, to the test tube containing magnetic bead detects liquid, vibrating of short duration centrifugation after mixing, (including nucleic acid expands The reagent and internal standard probe of increasing).
PCR temperature controls：45℃10min；
95℃10min；
95 DEG C of 15s, 60 DEG C of 45s；40 cycles；
Fluorescence selects bis- channels FAM, CY5.
As a result referring to Fig. 1, from figure one as can be seen that negative control and positive control are normal in Fig. 1, positive sample is detected as The positive, 2 negative samples are detected as negative (internal control is normal).
Examples of implementation 2：Specificity
According to examples of implementation 1 magnetic bead method for extracting nucleic acid to following sample carry out nucleic acid extraction, PCR amplification system and Amplification condition is identical as examples of implementation 1 (positive and negative control), and it is respectively influenza A virus to take 5 parts of specific reference materials (National reference), influenza B virus (National reference), Escherichia coli (ATCC35150), staphylococcus aureus (ATCC6538) and Candida albicans (ATCC10231) positive sample and RSV positive samples, fluorescent PCR kit is carried out Specific test.The sample of specificity is that positive sample and sample above mix to extract nucleic acid, and the method for nucleic acid extraction is the same as real It applies that example 1 is identical, then utilizes the amplification system of the present invention to carry out the amplification of nucleic acid, obtain the result such as Fig. 2.
As a result it shows (such as Fig. 2), shows that only positive control curve is shown as positive, and influenza A virus (country's reference Product), influenza B virus (National reference), Escherichia coli (ATCC35150), staphylococcus aureus (ATCC6538) and white Color candida albicans (ATCC10231) positive sample does not occur specific amplification curve, illustrates the pertussis nucleic acid detection reagent Box and influenza A virus (National reference), influenza B virus (National reference), Escherichia coli, staphylococcus aureus With Candida albicans no cross reaction.
Examples of implementation 3：Sensitivity
To with a concentration of 106CFU/ml pertussis Bao Te bacillus ATCC9430 samples are diluted to obtain 104CFU/ml、 103CFU/ml and 102CFU/ml, 101Then kit operation side that CFU/ml is referred to according to the method and reagent of examples of implementation 1 Formula carries out PCR amplification, and respectively detection is primary, and testing result is shown in Fig. 3.
Fig. 3 shows that the kit can detect a concentration of 104CFU/ml、103CFU/ml and 102CFU/ml、101CFU/ The Bordetella pertussis of ml shows that the fluorescence quantifying PCR method minimum detection limit of the kit is about 100-10copies/ul, has Preferable sensitivity resolution ratio.Being compared than traditional cultivation and antigen detection method using this method is had quickly, accurately, spirit The high feature of sensitivity.
The all patents and publications mentioned in description of the invention all indicates that these are the public technology of this field, this hair It is bright to use.All patents referred to herein and publication are all equally listed in bibliography, with each publication It is specific to be individually referenced equally.The present invention described here can lack any type element or multiple element, and one It is realized in the case of kind limitation or a variety of limitations, this limitation here is not particularly illustrated.Such as art in each example here Language "comprising", " essence by ... form " and " by ... form " can be replaced with remaining 2 term of one of both.Here it adopts Describing mode carried out by terms and expressions mode, and be not limited except as, also indicate that this book describes without any intention here These terms and explain and eliminate any equivalent feature, but it is recognised that can be in the model of the present invention and claim Any suitable be altered or modified is done in enclosing.Preferably implement it is appreciated that examples of implementation described in the invention are all some Example and feature, any those of ordinary skill in the art can be changed and become according to some are done under the marrow of the invention that describe Change, these are changed and variation is recognized as belonging to the scope of the present invention and independent claims and appended claims are limited In the range of.

Claims (9)

1. the kit for detecting nucleic acid of Bordetella pertussis in a kind of detection sample, which includes nucleic acid cleavage solution, wherein Nucleic acid cleavage solution includes magnetic bead, and the nucleic acid cleavage solution includes 1M NaCl, 0.01%Triton-X and 0.001M KCl, the hydroxyl magnetic bead that magnetic bead is 0.005 milligram.
2. a kind of detection Bordetella pertussis kit for detecting nucleic acid, which includes lysate and bead suspension, wherein is split The ingredient for solving liquid includes 1M NaCl, 0.01%Triton-X and 0.001M KCl, and bead suspension includes the magnetic of hydroxyl modified Pearl, polydispersity coefficient ＜ 0.2；Magnetic bead is that diameter is less than or equal to 500nM, wherein the content of magnetic bead is 10mg/mL.
3. according to the kit described in one of claim 1-2, wherein after lysate and magnetic bead solution mix, the body of the two Product is than being 200:1.
4. according to the kit described in one of claim 1-3, wherein the reagent further includes a kind of mixture, which is Solution mixture, the mixture include cracking ingredient and magnetic bead, are divided into 1M NaCl, 0.01%Triton-X wherein being cracked into With 0.001M KCl, the hydroxyl magnetic bead for the 10mg/mL that magnetic bead is 0.005 microlitre.
5. according to the kit described in one of claim 1-4, wherein the kit further includes that the institute of PCR reaction amplifications is necessary Ingredient, the amplifing reagent includes the Mg2+ of 1~10mmol/L, the Tris-HCl buffer solutions of 10~50mmol/L；50~ The DNTP of 1000umol/L；The K ions of concentration 25mmol/L, the enzyme stability reagent of 100 μ g/ml, such as BSA etc.；Glycerine and Such as SEQ.1.1-1.3；Primer sequence and probe sequence shown in SEQ.2.1-2.3 and SEQ.3.1-3.3.
6. according to the kit described in one of claim 1-5, wherein the kit further includes：Negative control, positive control, say Bright book and box body composition.
7. according to the kit described in one of claim 1-6, wherein the sample be serum, urine, mouthwash, hydrothorax, Ascites, cerebrospinal fluid or aqueous humor equal samples, the sample are used directly for nucleic acid extraction；Alternatively, swab sample, such as nasopharyngeal swab Son, sputum class sample, wherein swab or sputum sample are dissolved in 1-5mL physiological saline.
8. the method for the nucleic acid of Bordetella pertussis, this method include in a kind of detection sample：
Kit as described in one of claim 1-7 is provided,
The extraction and amplification of sample of nucleic acid, wherein the method for the nucleic acid extraction is as follows：
(1), sample is allowed to be contacted in PCR pipe with magnetic bead lysate, wherein the volume ratio of magnetic bead lysate and sample is 4:1, In, sample is 20 microlitres；
(2), 60-100 DEG C is heated to the PCR pipe of step (1), 10min is placed at room temperature for after 5min；
(3), the liquid in PCR pipe is removed, only retains magnetic bead, while any subsequent processing is not done to magnetic bead；
(4), the necessary reagent of nucleic acid is added into PCR pipe, such reagent includes such as SEQ.1.1-1.3 and SEQ.2.1-2.3 Shown in primer sequence and probe sequence；
(5), the amplification of at least one cycles of PCR is carried out.
9. according to the method described in claim 8, the sample be serum, urine, mouthwash, hydrothorax, ascites, cerebrospinal fluid or Person's aqueous humor equal samples, the sample are used directly for nucleic acid extraction；Alternatively, swab sample, such as Nasopharyngeal swabs, sputum class sample This, wherein swab or sputum sample is dissolved in 1-5mL physiological saline.
CN201810172491.6A 2017-03-03 2018-03-01 One-step method detects the kit and detection method of pertussis nucleic acid Pending CN108486259A (en)

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CN201710122152.2A CN106636446A (en) 2017-03-03 2017-03-03 Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample
CN2017101221522 2017-03-03

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CN201710122152.2A Pending CN106636446A (en) 2017-03-03 2017-03-03 Direct real-time quantitative PCR method of throat swab sample or nasopharyngeal swab sample
CN201810172492.0A Active CN108411036B (en) 2017-03-03 2018-03-01 Nucleic acid detection kit and method for rapidly detecting influenza A and influenza B viruses
CN201810172541.0A Active CN108441580B (en) 2017-03-03 2018-03-01 Kit for simultaneously detecting HSV-1, HSV-2, VZV herpes viruses by one-step method and detection method
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