CN106337085A - Nucleic acid test card and application method thereof - Google Patents
Nucleic acid test card and application method thereof Download PDFInfo
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- CN106337085A CN106337085A CN201610710500.3A CN201610710500A CN106337085A CN 106337085 A CN106337085 A CN 106337085A CN 201610710500 A CN201610710500 A CN 201610710500A CN 106337085 A CN106337085 A CN 106337085A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention provides a nucleic acid test card and an application method thereof. The nucleic acid test card comprises a sealing film, a card body, a reagent hole, a nucleic acid amplification reagent, reaction holes, nucleic acid collection test paper, a nucleic acid purifying reagent for matching use, a washing buffer and an organic phase for preventing a solution from being volatized. The application method comprises the following steps: opening the sealing film, adding a pathogene sample into the reaction holes packaged with the nucleic acid collection test paper, automatically washing by the use of the nucleic acid purifying reagent and the washing buffer for several times, respectively adding the nucleic acid amplification reagent in the reagent hole into each corresponding reaction hole to carry out a nucleic acid amplification reaction under the protection of the organic phase for preventing a solution from being volatized, and carrying out qualitative and quantitative analysis on the reaction result through an optical inspection instrument for matching use. By the adoption of the test card and through a nucleic acid amplification reaction, simultaneous testing of a single sample or various pathogens or target genes in various samples is realized. Time consumption of the test is low, test accuracy and sensitivity are high. The test card is applicable in the fields of experiment research, clinical test and food safety testing, etc.
Description
Technical field
The present invention relates to foranalysis of nucleic acids detection field, specifically a kind of detection of nucleic acids card based on nucleic acid amplification reaction
And its using method.
Background technology
, in the outburst of most of China area, the epidemic spreads of bird flu in 2005 and 2014 are non-for sars in 2003
Ebola's epidemic situation of high fatality rate is wreaked havoc in continent, and this is a series of to be taught that in the development history of the mankind by the infectious disease that virus causes
It is constantly subjected to the mortality threat that virus is brought.The propagating and the variation well beyond mankind's it is expected that timely and accurately of virus
Diagnosis virus, for improving patient survival, controls epidemic situation to propagate and effective disease treatment has very great meaning.
Conventional the pathogenic microorganism examination method is to adopt Vitro Culture Techniques, but, only about 1% antibacterial or virus
Can cultivate, and the complexity of incubation and very long incubation time, greatly limit the popularization of such technology.With point
The biological development of son, detection of nucleic acids is applied among Viral diagnosis, not only shortens detection time and also improves detection
Sensitivity and specificity.Nucleic acid molecules are heredity carriers including virus for all pathogen, by specific nucleic acid
Detected it can be determined that whether there is virus accordingly in sample.Polymerase chain reaction (pcr) is modal nucleic acid inspection
Survey method, the appearance of this technology makes the medical science of nucleic acid level detect and enters a brand-new stage.Pcr technology is passed through in difference
The duplication of dna molecule is realized in circulation between temperature, and this technology needs to temperature precise control, therefore depends on the temperature control of precision
The personnel of device and specialty are operated.In resource deficient area relative with manpower, pcr method is to be difficult to use in Viral diagnosis
's.In order to overcome the dependence to attemperating unit, a series of isothermal amplification methods arise at the historic moment, such as ring mediated isothermal amplification
(lamp) etc..Ring mediated isothermal amplification can expand to target nucleic acid at 60-65 DEG C.Isothermal amplification method not only can
It is a kind of quick, sensitive, accurate detection of nucleic acids mode, temperature control and testing equipment are required simple simultaneously, significantly reduce and set
The input of standby cost.Optical detection or the Turbidity measurement based on magnesium pyrophosphate by-product are coordinated using dna fluorescence indicator, can
Quantitative analyses are carried out to sample.
In clinical case, same disease may be produced by multiple different pathogen, cause diagnosis and suit the medicine to the illness
The difficulty write a prescription.Using nucleic acid amplification method, either pcr or isothermal duplication, how to detect simultaneously multiple pathogens or
Target gene is always one of Clinical detection significant challenge.Therefore, invention one kind is simple to operate, accuracy is high, sensitivity
High detection of nucleic acids product, realizes detecting while multiple pathogens or target gene significant.
Content of the invention
It is an object of the invention to provide a kind of detection of nucleic acids card and its using method, there is easy to carry, simple to operate, sample
Product consumption is few, can automatization the features such as.
A kind of detection of nucleic acids card, collects reagent paper including sealed membrane, card body, nucleic acid, and matching used nucleic acid purification reagent,
Dcq buffer liquid, described card body is provided with multiple reagent wells and reacting hole, and described reagent in the hole is preinstalled with nucleic acid amplification agents, institute
State reaction in the hole and be fixed with nucleic acid collection reagent paper.
Described sealing membrane material is plastics, is fixed on detection of nucleic acids card by viscose glue or electrostatic interaction, for sealing
State reagent wells and described reacting hole.
Described card body material can be metal, glass or plastics.
Described nucleic acid amplification agents include enzyme needed for primer, nucleic acid amplification reaction, reaction buffer, triphosphoric acid deoxidation core
Thuja acid and visible ray or fluorescence indicator.
The corresponding nucleic acid amplification reaction of described nucleic acid amplification agents, can be anti-for the polymerase chain expanding template with dna
Should (pcr), loop-mediated isothermal amplification (lamp), rolling circle amplification reaction (rca), the isothermal amplification of dependence unwindase
(hda), the isothermal amplification (rpa) of recombinase-mediated, with rna for expand template RT-polymerase chain reaction (rt-
Pcr), one of reverse transcription loop-mediated isothermal amplified reaction (rt-lamp) or other reverse transcription nucleic acid amplification reactions.
Described reacting hole is arranged in an array manner, in addition to the reacting hole for sample detection, it is possibility to have as sun
Property and the reacting hole of negative control.
Described nucleic acid collects the chemical substance that reagent paper contains uniqueness, can make protein denaturation effectively with cell lysis
Protect nucleic acid from nuclease, oxidant and ultraviolet injury.
Described organic faciess be pre-installed on detection of nucleic acids card beyond test tube container in, its density be less than 1 gram every milliliter.
Described nucleic acid purification reagent and dcq buffer liquid be pre-installed on detection of nucleic acids card beyond the container such as test tube in.
The using method of detection of nucleic acids card of the present invention be sealed membrane is taken off from card body after, detection of nucleic acids card is placed on
The detection card memory element of matching used nucleic acid amplification detector, and sample-adding and sample are completed by robotic
Process: sample to be detected adds reacting hole, collect reagent paper with nucleic acid and react, after incubation a period of time, add nucleic acid purification reagent
Rinse, remove nucleic acid purification reagent, add dcq buffer liquid to rinse, remove dcq buffer liquid, the core in reagent wells will be pre-installed on
Sour amplifing reagent is transferred in reacting hole, adds the organic faciess preventing solution evaporation in reacting hole, according to detection object with instead
Answer the difference of system, nucleic acid amplification reaction is realized by suitable temperature control, and using suitable wave band in reacting hole
Reaction system carries out optical detection.
The technique effect of the present invention is embodied in:
Multiple nucleic acid amplification reaction system are pre-stored in detection of nucleic acids card the present invention, by matching used nucleic acid amplification
Detector automatization completes sample introduction, sample treatment, nucleic acid amplification, optical detection etc., realizes to single sample or multisample
In multiple pathogens or target gene detected simultaneously；The result of nucleic acid amplification can by optical detection carry out qualitative with
Quantitative analyses.
Brief description
Fig. 1 is apparatus of the present invention schematic diagram.
Fig. 2 is to carry out specific nucleic acid testing result figure using this detection of nucleic acids card.
Specific embodiment
Below in conjunction with the accompanying drawings the present invention is done and further describe in detail.
Detection of nucleic acids card of the present invention, as shown in Figure 1, comprising: sealed membrane 1, card body 2, reagent wells 3, nucleic acid amplification
Reagent 4, reacting hole 5, nucleic acid are collected reagent paper 6, matching used nucleic acid purification reagent 7, dcq buffer liquid 8, are prevented solution evaporation
Organic faciess 9 and nucleic acid amplification detector.Nucleic acid amplification agents 4 are pre-installed in reagent wells 3.It is solid in advance that nucleic acid collects reagent paper 6
In reacting hole 5.After sealed membrane 1 takes off from card body 2, detection of nucleic acids card is placed on matching used nucleic acid amplification inspection
Survey the detection card memory element of instrument, and following work are completed by robotic: sample to be detected adds reacting hole 5, with
Nucleic acid is collected reagent paper 6 and is reacted, and after incubation a period of time, adds nucleic acid purification reagent 7 to rinse, removes nucleic acid purification reagent 7, add
Dcq buffer liquid 8 rinses, and removes dcq buffer liquid 8, the nucleic acid amplification agents 4 being pre-installed in reagent wells 3 are transferred to reacting hole 5
In, add the organic faciess 9 preventing solution evaporation in reacting hole 5.Nucleic acid amplification reaction is realized by temperature control, and using conjunction
Suitable wave band carries out optical detection to the reaction system in reacting hole 5.The reacting hole 5 of array can achieve inspection while multisample
Survey.Additionally, the nucleic acid amplification agents 4 in different reagent wells 3 comprise different primer pairs, can achieve to multiple pathogens or mesh
Detection while mark gene.
Below by a specific embodiment, the present invention is described further, but specific embodiment is not to this
Any restriction is done in invention.
Example 1: detect while multiple intestinal bacteria.
Prepare the detection of nucleic acids card that can be used for that multiple intestinal bacteria detect simultaneously first:
1) make the detection of nucleic acids card based on plastic material.4 reagent wells, pre-install ring mediated isothermal amplification reagent respectively,
Including primer (being respectively used for amplifying the specific target gene of escherichia coli, Salmonella, dysentery bacterium, vibrio parahaemolytious), bst
Polymerase, tris hydrochloride buffer, triphosphate deoxy-nucleotide, calcium yellow-green fluorescence indicator；4 × 5 reacting holes, each is anti-
Ying Kongzhong fixes one block of nucleic acid and collects reagent paper (such as whatman fta reagent paper), for 3 samples, 1 negative control and 1
Detect while positive control；
2) adopt adhesive closure film to seal reagent wells and reacting hole, and preserve detection of nucleic acids card in -20 DEG C long-term.
The detection of nucleic acids card preparing can be examined to escherichia coli, Salmonella, dysentery bacterium, vibrio parahaemolytious simultaneously
Survey:
1) people's Excreta is put into sample tube, and add appropriate tris hydrochloride buffer；
2) it is loaded: sample cell is put into nucleic acid amplification detector, a detection of nucleic acids card is put into nucleic acid amplification inspection simultaneously
Survey the detection card memory element of instrument, 3 samples, 1 negative control and 1 positive control are added reacting hole by mechanical hand automatically
In, 4 reacting holes of each sample；
3) sample treatment: sample reacts after about 1 minute with the nucleic acid extraction reagent paper in reacting hole, mechanical hand is automatically toward each anti-
Ying Kongzhong adds nucleic acid purification reagent to rinse once, is subsequently adding te wash buffer once, and removes all reagent；
3) react: mechanical hand automatically the nucleic acid amplification agents in reagent wells is transferred in reacting hole, every kind of reagent 5 is anti-
Ying Kong, and in each reacting hole, addition prevents the mineral oil organic faciess of solution evaporation, by temperature control, in 65 degrees Celsius of realities
Existing nucleic acid amplification reaction；
4) detect: using matching used fluorescence detector automatic detection reaction result, (470 nanometers excite, 520 nanometers
Detection), it is illustrated in figure 2 E. coli detection result, positive hole 10 fluorescence is stronger, and negative hole 11 fluorescence is weaker, a sample
Hole 12 fluorescence is weaker, and No. two sample well 13 fluorescence are stronger, and No. three sample well 14 fluorescence are stronger.Can be straight according to fluoroscopic examination result
Connect and judge to comprise escherichia coli in No. two and No. three samples.Other bacterial strain detection methods are similar to.
As it will be easily appreciated by one skilled in the art that the foregoing is only presently preferred embodiments of the present invention, not in order to
Limit the present invention, all any modification, equivalent and improvement made within the spirit and principles in the present invention etc., all should comprise
Within protection scope of the present invention.
Claims (9)
1. a kind of detection of nucleic acids card it is characterised in that: include sealed membrane (1), card body (2), nucleic acid collect reagent paper (6), and supporting
The nucleic acid purification reagent (7) that uses, dcq buffer liquid (8), organic faciess (9), described card body (2) is provided with reagent wells (3) and instead
Answer hole (5), in described reagent wells (3), be preinstalled with nucleic acid amplification agents (4), be fixed with nucleic acid in described reacting hole (5) and collect examination
Paper (6).
2. detection of nucleic acids card according to claim 1 is it is characterised in that described sealed membrane (1) material is plastics, by viscous
Glue or electrostatic interaction are fixed on detection of nucleic acids card, for sealing described reagent wells (3) and described reacting hole (5).
3. detection of nucleic acids card according to claim 1 is it is characterised in that described card body (2) material can be metal, glass
Or plastics.
4. detection of nucleic acids card according to claim 1 is it is characterised in that described nucleic acid amplification agents (4) include primer, core
Enzyme needed for sour amplified reaction, reaction buffer, triphosphate deoxy-nucleotide and visible ray or fluorescence indicator.
5. detection of nucleic acids card according to claim 1 is it is characterised in that the corresponding nucleic acid of described nucleic acid amplification agents (4)
Amplified reaction, can be with dna for expand the polymerase chain reaction of template, loop-mediated isothermal amplification, rolling circle amplification reaction,
Rely on the isothermal amplification of unwindase, the isothermal amplification of recombinase-mediated, with rna for expanding the Reverse Transcription Polymerase of template
One of enzyme chain reaction, reverse transcription loop-mediated isothermal amplified reaction or other reverse transcription nucleic acid amplification reactions.
6. detection of nucleic acids card according to claim 1 is it is characterised in that described reacting hole (5) is arranged in an array manner.
7. detection of nucleic acids card according to claim 1 contains uniqueness it is characterised in that described nucleic acid collects reagent paper (6)
Chemical substance, can be made protein denaturation and is effectively protected nucleic acid from nuclease, oxidant and ultraviolet line loss with cell lysis
Wound.
8. detection of nucleic acids card according to claim 1 is it is characterised in that described organic faciess (9) are pre-installed on detection of nucleic acids card
In test tube container in addition, its density is less than 1 gram every milliliter.
9. a kind of using method of detection of nucleic acids card as claimed in claim 1 it is characterised in that: by sealed membrane (1) from card body
(2), after taking off on, detection of nucleic acids card is placed on the detection card memory element of matching used nucleic acid amplification detector, and passes through
Robotic completes following work: sample to be detected adds reacting hole (5), collects reagent paper (6) with nucleic acid and reacts, is incubated one
After the section time, add nucleic acid purification reagent (7) to rinse, remove nucleic acid purification reagent (7), add dcq buffer liquid (8) to rinse, remove
Remove dcq buffer liquid (8), the nucleic acid amplification agents (4) being pre-installed in reagent wells (3) are transferred in reacting hole (5), add anti-
Only the organic faciess (9) of solution evaporation, in reacting hole (5), realize nucleic acid amplification reaction by temperature control, and using suitable
Wave band carries out optical detection to the reaction system in reacting hole (5).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610710500.3A CN106337085A (en) | 2016-08-24 | 2016-08-24 | Nucleic acid test card and application method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610710500.3A CN106337085A (en) | 2016-08-24 | 2016-08-24 | Nucleic acid test card and application method thereof |
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| Publication Number | Publication Date |
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| CN201610710500.3A Pending CN106337085A (en) | 2016-08-24 | 2016-08-24 | Nucleic acid test card and application method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107385103A (en) * | 2017-09-18 | 2017-11-24 | 星源智（珠海）生物科技有限公司 | A kind of non-purification of nucleic acid amplification method and device |
| CN107513494A (en) * | 2017-08-17 | 2017-12-26 | 湖北伽诺美生物科技有限公司 | A kind of detection of nucleic acids card and its application method |
| CN108949548A (en) * | 2018-09-14 | 2018-12-07 | 昆明医科大学第附属医院 | A kind of full-automatic nucleic acid amplification assays instrument based on isothermal amplification reactions |
| CN110218769A (en) * | 2019-07-08 | 2019-09-10 | 中国人民解放军东部战区疾病预防控制中心 | The multi-joint test strips of integration integral type for LAMP or RT-LAMP |
| CN110358666A (en) * | 2019-06-28 | 2019-10-22 | 马鞍山众慧惠生生物科技有限公司 | Box integrated nucleic acid quantification detection device |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107513494A (en) * | 2017-08-17 | 2017-12-26 | 湖北伽诺美生物科技有限公司 | A kind of detection of nucleic acids card and its application method |
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| CN108949548A (en) * | 2018-09-14 | 2018-12-07 | 昆明医科大学第附属医院 | A kind of full-automatic nucleic acid amplification assays instrument based on isothermal amplification reactions |
| CN110358666A (en) * | 2019-06-28 | 2019-10-22 | 马鞍山众慧惠生生物科技有限公司 | Box integrated nucleic acid quantification detection device |
| CN110218769A (en) * | 2019-07-08 | 2019-09-10 | 中国人民解放军东部战区疾病预防控制中心 | The multi-joint test strips of integration integral type for LAMP or RT-LAMP |
| CN110218769B (en) * | 2019-07-08 | 2024-04-19 | 中国人民解放军东部战区疾病预防控制中心 | Integrated multi-linked test strip for LAMP or RT-LAMP |
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Application publication date: 20170118 |