CN106244582A - A kind of buccal swab nucleic acid room temperature store method - Google Patents
A kind of buccal swab nucleic acid room temperature store method Download PDFInfo
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- CN106244582A CN106244582A CN201610749927.4A CN201610749927A CN106244582A CN 106244582 A CN106244582 A CN 106244582A CN 201610749927 A CN201610749927 A CN 201610749927A CN 106244582 A CN106244582 A CN 106244582A
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Abstract
The present invention relates to technical field of biological, disclose a kind of buccal swab nucleic acid room temperature store method, contain two steps, the first step is to detect the quality of buccal swab nucleic acid to be saved, second step is that the room temperature carrying out buccal swab nucleic acid preserves, on the premise of guaranteeing the Nucleic acid quality needing to preserve, the multiple qualificationss preserving room temperature are tested, have selected the actual conditions of preservation, advantages of the present invention be simple to operate, consider comprehensively, control the temperature range of preservation and the time span of preservation, can preferably be applied in practical situation.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of buccal swab nucleic acid preservation method.
Background technology
Buccal swab is nucleic acid source important in molecular epidemiology, hereditism and prudence research, and its collection has
Sampling simplicity, safety, low cost, AT advantage.The cold strike of nucleic acid is made it can not obtain after being saved by cryopreservation
Preferably preservation effect, High temperature storage nucleic acid can make again energy metabolism aggravation, the reproduction speed of antibacterial is accelerated, and causes nucleic acid short
The decline phenomena of mortality are just showed in time.Show that the optimal storage temperature of buccal swab nucleic acid is at 25 DEG C-45 DEG C through research., and
Store method poor effect now, accordingly, it would be desirable to a kind of store method can meet the long purpose of room temperature.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of room temperature, long buccal swab nucleic acid is protected
Deposit method；
Present disclosure is: a kind of buccal swab nucleic acid room temperature store method, it is characterised in that can be at 25 degree to 45
Preserve 6 months under the room temperature of degree, specifically comprise the following steps that
The first step, detects the quality of buccal swab nucleic acid to be saved；
Buccal swab nucleic acid samples percentage of head rice and abnormal rate detection, first prepare the wave carrier piece of cleaning, clean with dish detergent, clearly
Water rinses, then rinses with distilled water, is clean not hang the globule, dries or dry standby, smear: shaken up by the sample that need to measure,
Take drip a middle level sample put down drip in the right-hand member of microscope slide, take the microscope slide that another the smooth of the edge is straight, in 35 ° of angles from buccal swab
The left side of nucleic acid samples contacts to the right sample, sample is i.e. distributed in strip between two microscope slide engagement edges, by above
Microscope slide againsts the slide surface of horizontal, and right-to-left moves, and is evenly distributed on microscope slide with sample, make
Smear back side right-hand member is numbered with extraordinary marking pen, and every part of sample need to make two smears, natural air drying 5～20min simultaneously.Gu
Fixed: air-dried smear is flat on staining rack, draw 1ml fixative with dropper, drip in smear, and make fixative be covered with in
Whole smear surface, and fixing 15min.Washing: live smear one end with object slide forceps sub-folder, fixative is discarded and pours dyeing into
In cylinder or plate, equipped with in the beaker of distilled water, rock up and down and rinse, clamp and unclamp tweezers several times, take out smear in
Disk limit, waits to do.Dyeing: the smear after fixing be flat on staining rack, draws new preparation Giemsa dye liquor about with dropper
2ml, is placed in microscope slide to be contaminated parallel above.Extrusion dye liquor is in smear from left to right, makes dye liquor uniformly be covered with in smear.
Static dyeing 90min.Washing: clamp dye sheet with tweezers, discards the dye liquor on dye sheet and pours in plate, then equipped with tap water
Beaker in rinse, purging method is ibid.Rinse through for several times, till water lotion is colourless.During washing, if water becomes
Indigo plant, then can change clear water in time.Disk limit can be stood on after Xi Jinging, wait to do.By dye sheet in detecting instrument, detect quality, the fullest
The quality of the foot buccal swab nucleic acid of definite quality in advance just can carry out next step room temperature and preserve, and this quality is that nucleic acid is in oral cavity
In swab, content is more than 65%；
Second, the room temperature carrying out buccal swab nucleic acid preserves；Diluent pH entire change, for falling before, rises, finally afterwards
Tend towards stability.Analyzing its reason is that cell nucleic acid is the most metabolic, can produce acidic metabolic in this course and produce
Thing, this acidic metabolite makes pH decline.Because diluent formulation has the bufferings such as sodium citrate, Tris, sodium bicarbonate
Agent, can balance nucleic acid metabolism product and maintain the most stable of nucleic acid living environment, play the effect of protection nucleic acid equally.Press respectively
Buccal swab nucleic acid and diluent ratio are 1: 1,1: 1.5,1: 21: 2.5,1: 3 five kind of dilution proportion, measure nucleic acid vigor and become
Change, to determine optimal dilution ratio.1: 1,1: 1.5, the preservation effect of 1: 2 three kind of ratio is good, 1: 1, and 1: 1.5,1: 2 three kind of ratio
In example, the preservation effect of 1: 1,1: 1.5 two kind of ratio is the best, and difference is not notable (P ＞ 0.05).The guarantor of 1: 2.5,1: 3 two kind of ratio
Deposit effect approximation (P ＞ 0.05), but not as above-mentioned three kinds.Consider, select 1: 1.5 diluent preserved as room temperature；
The interpolation of antibiotic is the emphasis during buccal swab nucleic acid room temperature preserves, and the effect of antibiotic is suppression dilution
In liquid, antibacterial grows, for spermatid offer health environment.The result of this test shows, the effect of penicillin is the most mould not as celebrating
Element, and the intermixture best results of penicillin+gentamycin, select this intermixture；
Temperature is to affect buccal swab nucleic acid room temperature to preserve the representative of extraneous factor, and getting up early is for buccal swab nucleic acid room temperature
In the research of the preference temperature preserved, it is believed that low temperature can suppress the activity of nucleic acid, thus reach to extend the holding time
Purpose.And substantial amounts of experimentation shows, low temperature is very big for the impact of buccal swab cell nucleic acid, can cause buccal swab core
The inactivation of acid cell, saving of the buccal swab that is the most just far from being nucleic acid.Recent research indicate that, under the conditions of 25 degree to 45 degree,
Preservation effect for buccal swab nucleic acid is best, and being wherein generally acknowledged 30 DEG C of room temperature conditions is that room temperature preserves best temperature
Degree.
Detailed description of the invention
In order to make the technical problem to be solved, technical scheme and beneficial effect clearer, the most right
The present invention is described in detail.It should be noted that, specific embodiment described herein is only in order to explain the present invention, not
For limiting the present invention, the product that can realize said function belongs to equivalent and improvement, is all contained in the protection model of the present invention
Within enclosing.Concrete grammar is as follows:
Diluent is the key factor affecting buccal swab nucleic acid room temperature preservation effect, and the Main Function of diluent is: one
Aspect is used for expanding nucleic acid volume to meet Production requirement；The opposing party and protect the functional character of nucleic acid.Contained by diluent
Various compositions are the most different to the protective effect of buccal swab nucleic acid, nucleic acid need produce energy with maintain cellular metabolism and from
Body moves.Glucose can not only provide energy for nucleic acid, it is also possible to maintains the osmotic pressure of diluent, makes in most formula
With.Peracid is crossed the environment of alkali and is all unfavorable for that nucleic acid is survived, and needs to add a certain amount of buffer substance to maintain nucleic acid in diluent
pH.Sodium bicarbonate or sodium citrate are often used to the buffer capacity of the buffer agents such as pH, Tris and the HEPES of regulation diluent more
By force.EDTA is a kind of chelating agen, it is possible to the especially Ca2+ of the metal ion in diluent is combined, and reduces entrance intracellular
Ca2+, prevents from inducing nucleic acid capacitation and the generation of acrosome reaction, to reach to suppress nucleic acid movable, increases the effect of nucleic acid preservation time
Really.Certain density+Na+-K+ pump operating on nucleic acid film can be maintained, but the K+ concentration in must assure that cell nucleic acid is higher than cell
Outer K concentration, so could produce ATP and maintain depositing and moving of nucleic acid, thus diluent can add appropriate KC1.It addition, it is dilute
Releasing interpolation antibiotic in liquid can suppress the breeding of antibacterial, main conventional antibiosis to have penicillin, gentamycin etc..But by
In modern society for the abuse of antibiotic so that a lot of antibacterials have obvious resistance for common antibiotics, so, novel
Antibiotic be applicable to buccal swab nucleic acid room temperature preserve research extremely the most urgent.
Buccal swab nucleic acid individually preserves, and harmful microorganism therein not only can fight for nutrient substance, and metabolism is produced
Raw toxic metabolite refuse can change Conservation environment, has a strong impact on preservation effect.Existing mushroom have escherichia coli, Salmonella
Bacterium, klebsiella, Bacillus proteus and corynebacterium etc..Gram negative bacteria is mushroom common in boar semen.Therefore dilute
Release to add in agent has the antibiotic of inhibition optimal to gram negative bacteria.In diluent, add gentamycin suppress
The growth of antibacterial, the ill effect to cell is little.The povidone iodine adding 0.2mg/ml in diluent can kill pathogenic thin
Bacterium, and semen at normal temperature is preserved quality index without impact.Have researcher the buccal swab of separate sources is carried out bacteria distribution,
Identify, cultivate and find during drug sensitive test, in five kinds of antibacterials of detection, the Kui Ruo such as ciprofloxacin, ofloxacin and norfloxacin
Ketone medicine is preferable to its inhibition, and finds that Bacillus proteus, corynebacterium and Salmonella are to penicillin and clindamycin
There is drug resistance.Comparing 7 antibiotic and combine the fungistatic effect of antibacterial in Buffalo Semen, result shows, penicillin, strepto-
Element and gentamycin all have an antibacterial circle, and that tylosin, cillimycin, neomycin and spectinomycin have in various degree is resistance to
Medicine phenomenon.The antibiotic combinations that the safe woods of celebrating is big in terms of antibiotic combinations is optimal.In actual application, the selection of antibiotic can be because of dilute
Release the difference of agent composition and different.
The invention has the beneficial effects as follows: advantages of the present invention be simple to operate, consider comprehensively, at room temperature buccal swab nucleic acid
Preservation in, control the temperature range of preservation and the time span of preservation, can preferably be applied in practical situation.
Claims (1)
1. a buccal swab nucleic acid room temperature store method, it is characterised in that 6 can be preserved under the room temperature of 25 degree to 45 degree
Month, specifically comprise the following steps that
The first step, detects the quality of buccal swab nucleic acid to be saved:
Buccal swab nucleic acid samples percentage of head rice and abnormal rate detection, first prepare the wave carrier piece of cleaning, clean with dish detergent, and clear water rushes
Wash, then rinse with distilled water, be clean not hang the globule, dry or dry standby, smear: the sample that need to measure is shaken up, take and drip
Middle level sample is put down and is dripped in the right-hand member of microscope slide, takes the microscope slide that another the smooth of the edge is straight, in 35 ° of angles from buccal swab nucleic acid
The left side of sample contacts to the right sample, and sample is i.e. distributed in strip between two microscope slide engagement edges, by load glass above
Sheet againsts the slide surface of horizontal, and right-to-left moves, and is evenly distributed on microscope slide with sample, in the smear made
Back side right-hand member is numbered with extraordinary marking pen, and every part of sample need to make two smears, natural air drying 5～20min simultaneously.Fixing: will
The smear air-dried is flat on staining rack, draws 1ml fixative with dropper, drips in smear, and makes fixative be covered with in whole
Smear surface, and fixing 15min.Washing: live smear one end with object slide forceps sub-folder, discards fixative and pours color jar or flat into
In ware, equipped with in the beaker of distilled water, rock up and down and rinse, clamp and unclamp tweezers several times, take out smear in disk limit,
Wait to do.Dyeing: the smear after fixing be flat on staining rack, draws new preparation Giemsa dye liquor about 2ml with dropper, is placed in and wants
The microscope slide of dye is parallel above.Extrusion dye liquor is in smear from left to right, makes dye liquor uniformly be covered with in smear.Static dyeing
90min.Washing: clamp dye sheet with tweezers, discards the dye liquor on dye sheet and pours in plate, then equipped with in the beaker of tap water
Rinsing, purging method is ibid.Rinse through for several times, till water lotion is colourless.During washing, if water has become blue, then can and
Time change clear water.Disk limit can be stood on after Xi Jinging, wait to do.By dye sheet in detecting instrument, detect quality, only meet and specify in advance
The quality of the buccal swab nucleic acid of quality just can carry out next step room temperature and preserve, and this quality is nucleic acid content in buccal swab
It is more than 65%；
Second step, the room temperature carrying out buccal swab nucleic acid preserves:
Diluent pH entire change, for falling before, rises afterwards, finally tends towards stability.Analyzing its reason is that cell nucleic acid is continuous
Metabolic, acidic metabolite can be produced in this course, this acidic metabolite makes pH decline.Because dilution
Agent prescription has the buffer agents such as sodium citrate, Tris, sodium bicarbonate, nucleic acid metabolism product can be balanced and maintain nucleic acid living environment
The most stable, play the effect of protection nucleic acid equally.It is 1: 1,1: 1.5,1 in buccal swab nucleic acid and diluent ratio respectively:
21: 2.5,1: 3 five kind of dilution proportion, measures nucleic acid vigour changes, to determine optimal dilution ratio.1: 1,1: 1.5,1: 2 three kind
The preservation effect of ratio is good, and 1: 1, in 1: 1.5,1: 2 three kind of ratio, the preservation effect of 1: 1,1: 1.5 two kind of ratio is the best, poor
Different not notable (P ＞ 0.05).Preservation effect approximation (P ＞ 0.05) of 1: 2.5,1: 3 two kind of ratio, but not as above-mentioned three kinds.Combine
Close and consider, select 1: 1.5 diluent preserved as room temperature；
The interpolation of antibiotic is the emphasis during buccal swab nucleic acid room temperature preserves, and the effect of antibiotic is in suppression diluent
Growing of antibacterial, provides health environment for spermatid.The result of this test shows, the effect of penicillin is not so good as gentamycin,
And the intermixture best results of penicillin+gentamycin, select this intermixture；
Temperature is to affect buccal swab nucleic acid room temperature to preserve the representative of extraneous factor, and getting up early preserves for buccal swab nucleic acid room temperature
Preference temperature research in, it is believed that low temperature can suppress the activity of nucleic acid, thus reach to extend the purpose of holding time.
And substantial amounts of experimentation shows, low temperature is very big for the impact of buccal swab cell nucleic acid, and buccal swab nucleic acid can be caused thin
The inactivation of born of the same parents, saving of the buccal swab that is the most just far from being nucleic acid.Recent research indicate that, under the conditions of 25 degree to 45 degree, for
The preservation effect of buccal swab nucleic acid is best, and being wherein generally acknowledged 30 DEG C of room temperature conditions is that semen at normal temperature preserves best temperature
Degree.
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| CN201610749927.4A CN106244582A (en) | 2016-08-30 | 2016-08-30 | A kind of buccal swab nucleic acid room temperature store method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645678A (en) * | 2016-12-21 | 2017-05-10 | 王乾 | Detection method of oral swab |
| CN107119044A (en) * | 2017-05-02 | 2017-09-01 | 重庆市公安局巴南区分局 | A kind of wiping reagent for contact DNA transfers on carrier |
| CN107254467A (en) * | 2017-07-14 | 2017-10-17 | 北京碳元科技有限公司 | Normal temperature preserves method of nucleic acid sample and products thereof and application method |
| CN107897171A (en) * | 2017-12-25 | 2018-04-13 | 上海捷瑞生物工程有限公司 | A kind of mouth swab sample preserves liquid and improves and apply |
-
2016
- 2016-08-30 CN CN201610749927.4A patent/CN106244582A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645678A (en) * | 2016-12-21 | 2017-05-10 | 王乾 | Detection method of oral swab |
| CN107119044A (en) * | 2017-05-02 | 2017-09-01 | 重庆市公安局巴南区分局 | A kind of wiping reagent for contact DNA transfers on carrier |
| CN107119044B (en) * | 2017-05-02 | 2020-11-06 | 重庆市公安局巴南区分局 | Wiping reagent for contact DNA transfer on carrier |
| CN107254467A (en) * | 2017-07-14 | 2017-10-17 | 北京碳元科技有限公司 | Normal temperature preserves method of nucleic acid sample and products thereof and application method |
| CN107897171A (en) * | 2017-12-25 | 2018-04-13 | 上海捷瑞生物工程有限公司 | A kind of mouth swab sample preserves liquid and improves and apply |
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