CN105695561A - Method for establishing quick acting internal control in two-stage polymerase chain reaction - Google Patents
Method for establishing quick acting internal control in two-stage polymerase chain reaction Download PDFInfo
- Publication number
- CN105695561A CN105695561A CN201410692880.3A CN201410692880A CN105695561A CN 105695561 A CN105695561 A CN 105695561A CN 201410692880 A CN201410692880 A CN 201410692880A CN 105695561 A CN105695561 A CN 105695561A
- Authority
- CN
- China
- Prior art keywords
- stage
- internal control
- nucleotide sequence
- polymerase chain
- chain reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for establishing quick acting internal control in a two-stage polymerase chain reaction. The method comprises the following steps: taking a nucleic acid sample, adding an internal control nucleic acid sequence, a first stage starter and an internal control nucleic acid starter to the nucleic acid sample, carrying out a first stage polymerase chain reaction to generate a first stage reaction sample, taking parts of the first stage reaction sample as an internal control test sample, testing the internal control nucleic acid sequence in the internal control test sample by using an internal control test paper to examine that whether amplification of the internal control nucleic acid sequence in the first stage polymerase chain reaction is successful or not, adding a second stage starter to the first stage reaction sample if an amplified internal control nucleic acid sequence is detected through the internal control test paper, and carrying out a second stage polymerase chain reaction to generate a second stage reaction sample.
Description
Technical field
The invention relates to a kind of method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex, refer in particular to a kind of addition internal control nucleotide sequence in first stage Polymerase Chain Reaction, and confirm internal control nucleotide sequence whether Successful amplification with reagent paper before second stage Polymerase Chain Reaction, and the situation causing Polymerase Chain Reaction failed containing Polymerase Chain Reaction inhibiting substances in nucleic acid samples can be detected ahead of time, to avoid producing under-referral result and carrying out non-essential second stage Polymerase Chain Reaction person。
Background technology
For reducing the infection that causes of tuberculosis with dead, Disease Control Departments of the U.S. in 2005 can jointly follow the main implementation work listed in " the specific objective tuberculin test of latent tuberculosis infection with control " specification and be with U.S.'s chest medicine in it: the inspection of latent tuberculosis infection, latent tuberculosis infection are controlled and three targets such as bed and experimental monitoring。
U.S.'s tuberculosis case increased by 20% from 1985 to 1992 years, and decline about 44% from 1993 to 2003 years new cases of tuberculosis, 14 are reduced to 2004,571 tuberculosis case circulars, new case circular number is continuous between 11 years of 1993 to 2004 years to decline, it is possible to gives the credit to utilization resource effectively and finds early, controls open tuberculosis disease sufferer and latent tuberculosis infection person。
Latent tuberculosis infection person is not all shown as mycobacterium tuberculosis positive without disease with disease, X-ray and bacteriology checking after referring to infection mycobacterium tuberculosis。Latent tuberculosis infected students can't infect other people, if but do not controlled, sick may send out into open tuberculosis patient, though open tuberculosis patient also can be cured under suitable doctor looks after in the future, if but tuberculosis sufferer is not properly controlled, it is possible to cause that it is dead and cause expansion to infect。
Many areas are dependent on tuberculin skin test for the diagnosis of latent infection case in the world, but at TaiWan, China, because implementing bacillus calmette-guerin vaccine injection policy for a long time, plus in the past high tuberculosis prevalence rate so that utilize tuberculin skin test diagnosis tuberculosis infection of hiding to have obstacles at TaiWan, China and be difficult to walk。Therefore, use molecule inspection technology such as Polymerase Chain Reaction (polymerasechainreaction, etc. PCR) method has proved to be the effective tool of tuberculosis infection sufferer of hiding for a large amount of screenings, and especially nido Polymerase Chain Reaction (nestedPCR) can increase the accuracy of detection m tuberculosis infection further。
But, utilize Polymerase Chain Reaction technology may be subject in a sputum corpse or other object for laboratory examination and chemical testing interference of protein mortifier or other polymerase inhibitors matter, cause the testing result of under-referral, affect the positive corpse or other object for laboratory examination and chemical testing recall rate of whole detection method, be therefore internal control should be set up in detection method and just can ensure that its usefulness。
Therefore, the present invention proposes a kind of method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex, internal control nucleotide sequence is added in nucleic acid samples, and in first stage Polymerase Chain Reaction, expand first stage target gene nucleotide sequence (if feminine gender is then absent from also will not being amplified) and internal control nucleotide sequence in the lump, and after first stage Polymerase Chain Reaction, internal control nucleotide sequence whether Successful amplification is confirmed with reagent paper before second stage Polymerase Chain Reaction, and the situation causing Polymerase Chain Reaction failed containing Polymerase Chain Reaction inhibiting substances in nucleic acid samples can be detected ahead of time between two-step polymerization enzyme chain reflex, to avoid producing under-referral result and carrying out non-essential second stage Polymerase Chain Reaction person, for the countries and regions in untapped or exploitation, can effectively alleviate its manpower and materials burden utilizing two-stage polymerisation enzyme chain reflex inspection disease。
Summary of the invention
The main purpose of the present invention, it is in that to provide a kind of method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex, it expands first stage target gene nucleotide sequence and internal control nucleotide sequence in first stage Polymerase Chain Reaction simultaneously, and after first stage Polymerase Chain Reaction, confirm internal control nucleotide sequence whether Successful amplification, therefore screening can be done sth. in advance before second stage Polymerase Chain Reaction and be likely to result in the nucleic acid samples of under-referral result containing Polymerase Chain Reaction inhibiting substances, and avoid carrying out non-essential second stage Polymerase Chain Reaction。
The secondary objective of the present invention, it is in that to provide a kind of method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex, it utilizes whether internal control test paper test internal control nucleotide sequence expands in first stage Polymerase Chain Reaction, it is simple to operate, can obtain the result of test in the short time to check the method for internal control nucleotide sequence with internal control test paper, therefore can reach the effect of quick-acting internal control。
In order to reach above-mentioned censured purpose and effect, present invention is disclosed a kind of method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex, its step comprises: obtain a nucleic acid samples；By an internal control nucleotide sequence, a first stage introduction to and an internal control nucleic acid introduction to adding to this nucleic acid samples, carry out a first stage Polymerase Chain Reaction, generate a first stage response sample；Taken out part as an internal control test sample by this first stage response sample, and utilize an internal control test paper to test this internal control test sample to check whether this first stage response sample comprises an amplification internal control nucleotide sequence；And if this internal control test paper records this amplification internal control nucleotide sequence, by a second stage introduction to adding to this first stage response sample, carrying out a second stage Polymerase Chain Reaction, generating a second stage response sample。
Accompanying drawing explanation
Fig. 1: it is the flow chart of steps of first embodiment of the invention；
Fig. 2: it is the internal control test paper structural representation of first embodiment of the invention；
Fig. 3 A: it is internal control test paper test result schematic diagram () of first embodiment of the invention；
Fig. 3 B: it is internal control test paper test result schematic diagram (two) of first embodiment of the invention；
Fig. 3 C: it is internal control test paper test result schematic diagram (three) of first embodiment of the invention；
Fig. 3 D: it is internal control test paper test result schematic diagram (four) of first embodiment of the invention；
Fig. 4: it is the internal control test paper test result figure of second embodiment of the invention；And
Fig. 5: it is the cold light readings result figure of second embodiment of the invention。
[figure number is to as directed]
1 internal control test paper
10 supporters
12 corpse or other object for laboratory examination and chemical testing layers
14 complex releasing layers
16 inspected layer
160 p-wires
162 control lines
18 water accepting layers
Detailed description of the invention
In order to the architectural feature making the present invention and effect of reaching have a better understanding and awareness, spy's preferred embodiment and coordinate detailed description, illustrate as follows:
A kind of method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex that the present invention discloses, its characteristic is in that: expand internal control nucleotide sequence in the lump in the first stage Polymerase Chain Reaction of two-stage polymerisation enzyme chain reflex, and after in the first stage, Polymerase Chain Reaction completes, utilize internal control test paper detection internal control nucleotide sequence whether Successful amplification in first stage Polymerase Chain Reaction, to decide whether to carry out second stage Polymerase Chain Reaction, the amplification if internal control nucleotide sequence fails, show nucleic acid sample is likely to have polymerase inhibitors matter to exist and causes the failure of first stage Polymerase Chain Reaction, therefore should not carry out second stage Polymerase Chain Reaction, to avoid the failure of second stage Polymerase Chain Reaction to produce the result of under-referral, the cost carrying out invalid second stage Polymerase Chain Reaction can be saved simultaneously。
First seeing also Fig. 1, it is the flow chart of steps of first embodiment of the invention；As it is shown in figure 1, the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex of the present invention comprises the steps of
Step S10: obtain nucleic acid samples；
Step S20: by internal control nucleotide sequence, first stage introduction to and internal control nucleic acid introduction to adding to nucleic acid samples；
Step S30: carry out first stage Polymerase Chain Reaction, generates first stage response sample；
Step S40: taken out part as internal control test sample by first stage response sample, and utilize internal control test paper test internal control test sample；
Step S50: whether internal control test paper can record amplification internal control nucleotide sequence；
Step S60: by second stage introduction to adding to first stage response sample；
Step S62: do not carry out second stage Polymerase Chain Reaction；And
Step S70: carry out second stage Polymerase Chain Reaction, generates second stage response sample。
The method built in two-stage polymerisation enzyme chain reflex by quick-acting internal controls of the present invention is applied to check the nido Polymerase Chain Reaction of mycobacterium tuberculosis by the present embodiment, nido Polymerase Chain Reaction belongs to two-stage polymerisation enzyme chain reflex, the first stage target gene nucleotide sequence that it expands in first stage Polymerase Chain Reaction is longer, the second stage target gene nucleotide sequence expanded in second stage Polymerase Chain Reaction is shorter and is contained in first stage target gene nucleotide sequence, after the method details of enforcement and actual result are specified in。
In step S10, the clinical corpse or other object for laboratory examination and chemical testing that place's reason testee obtains is to obtain a nucleic acid samples, the mycobacterium tuberculosis of detection it is intended in the present embodiment, about 5 milliliters of sputum corpse or other object for laboratory examination and chemical testing should be collected (if not immediately treating by testee, 4 DEG C of refrigerators should be deposited in), and add sodium hydroxide citrate left-handed acetyl cysteine (NaOH-citrate-NALC) solution with the ratio with corpse or other object for laboratory examination and chemical testing volume 1:1, process a sputum corpse or other object for laboratory examination and chemical testing to obtain sputum corpse or other object for laboratory examination and chemical testing precipitate。
The configuration proportion of NaOH-citrate-NALC solution is as follows: 1 gram of left-handed acetyl cysteine (N-acetyl-L-cysteine, NALC) powder is dissolved in the mixed solution of 50 milliliter of 8% sodium hydroxide, 50 milliliter of 5.88% sodium citrate and 100 milliliter of 20% sodium lauryl sulphate (sodiumdodecylsulfate, SDS)。
A sputum corpse or other object for laboratory examination and chemical testing is placed in 50 milliliters of centrifuge tubes, and add and the isopyknic NaOH-citrate-NALC solution of a sputum corpse or other object for laboratory examination and chemical testing, after shaking 30 seconds, upset more than 10 times repeatedly, so that NaOH-citrate-NALC solution is mixed homogeneously with a sputum corpse or other object for laboratory examination and chemical testing, NaOH-citrate-NALC solution and a sputum corpse or other object for laboratory examination and chemical testing within 15 minutes, is made fully to react then at left at room temperature, it is subsequently added phosphate buffered solution (phosphatebufferedsaline, PBS) to 50 milliliters of scales, with 3, the centrifugal force of 800 × g is after 15 minutes, remove supernatant, precipitate is then with 1 milliliter of PBS back dissolving, and add the secondary water of 10 milliliters, again with 3 after shaking 20 seconds, the centrifugal force of 800 × g 15 minutes, remove supernatant, namely sputum corpse or other object for laboratory examination and chemical testing precipitate is obtained (if not carrying out DNA extraction immediately, should by deposit in-70 DEG C of refrigerators)。
The sputum corpse or other object for laboratory examination and chemical testing precipitate of about 1 milliliter is moved to cracking test tube (lysistube), with 14, the centrifugal force of 000 × g 2 minutes, 25 microlitre strong basicities cracking buffer solution (lysisbuffer-1) are added after removing supernatant, shake 1 minute to fill part mixing sputum corpse or other object for laboratory examination and chemical testing precipitate and cracking buffer solution, cracking buffer solution and sputum corpse or other object for laboratory examination and chemical testing precipitate within 10 minutes, is made fully to react then at left at room temperature, subsequently this cracking test tube is heated 20 minutes with the dry bath device being preheated to 100 DEG C, the invisible spectro steam condensation of cracking is made with room temperature after cooling down 5 minutes, add 20 microlitre highly acids cracking buffer solution (lysisbuffer-2) and 15 microlitres 10 milli mole concentration (mM) trishydroxymethylaminomethane hydrochloric acid (Tris-HCl) solution (acid-base value 8.0), shake 10 seconds so that cracking invisible spectro mixed solution and precipitate Homogeneous phase mixing, subsequently with 14, the centrifugal force of 000 × g is after 2 minutes, the supernatant drawing 50 microlitres carefully moves to microcentrifugal tube preservation, namely (subsequent survey step only needs to take 5 microlitres to obtain this nucleic acid samples, this nucleic acid samples untapped should deposit in-70 DEG C of refrigerators)。
In step S20, by an internal control nucleotide sequence, a first stage introduction to and an internal control nucleic acid introduction to each 0.2 milli mole concentration (μM) reaction density add this nucleic acid samples, to expand first stage target gene nucleotide sequence (about 245 base pair) and this internal control nucleotide sequence (about 119 base pair) in follow-up this first stage Polymerase Chain Reaction carried out in the lump, this first stage introduction is to being used for expanding this first stage target gene nucleotide sequence, and this internal control nucleic acid introduction is to being used for expanding this internal control nucleotide sequence。
During enforcement, it is placed in PCR (platoon) microcentrifugal tube by this nucleic acid samples obtained in step S10 takes out 5 microlitres, and adds this internal control nucleotide sequence (using the nucleotide sequence of SEQIDNO.1 in the present embodiment), this first stage introduction to (using the nucleotide sequence of SEQIDNO.2 and SEQIDNO.3 in the present embodiment) and this internal control nucleic acid introduction to (in the present embodiment the nucleotide sequence of use SEQIDNO.4 and SEQIDNO.5) with the reaction density of each 1 skin mole concentration (pM)。
For convenience of checking this internal control nucleotide sequence in subsequent step, this internal control nucleic acid introduction is to being connected to an antigen and a part respectively at its 5 ' end, with this internal control nucleotide sequence of labelling two strands, with Digitoxin (digoxigenin in the present embodiment, DIG) as this antigenic mark SEQIDNO.4, and using biotin (biotin) as this ligand-labeled SEQIDNO.5。
This first stage target gene nucleotide sequence should comprise a second stage target gene nucleotide sequence, using in the follow-up second stage Polymerase Chain Reaction carried out as this second stage target gene nucleotide sequence of template amplification, in the present embodiment, this second stage target gene nucleotide sequence should be the specific sequence in M. tuberculosis genosome, and this second stage target gene nucleotide sequence can be checked after completing this first stage Polymerase Chain Reaction and this second stage Polymerase Chain Reaction to judge whether there is M. tuberculosis in the sputum corpse or other object for laboratory examination and chemical testing of testee。
In step S20,0.2 microlitre deoxyribonucleic acid polymerase (DNApolymerase should be added further in PCR (platoon) microcentrifugal tube, generally use Taq), four kinds of deoxyribose triphosphoric acid (Deoxynucleotidetriphosphates of 44.8 microliters polymerase reaction buffers and mixing that reaction density is each 0.2 micro-mole concentration, dNTPs), to carry out this first stage Polymerase Chain Reaction in step S30。
In step S30, carry out this first stage Polymerase Chain Reaction to expand this first stage target gene nucleotide sequence and this internal control nucleotide sequence, generate a first stage response sample。In the present embodiment, in this first stage Polymerase Chain Reaction, reaction condition is as follows:
In step S40, an internal control test sample is obtained by this first stage response sample completing the generation of this first stage Polymerase Chain Reaction, and utilize an internal control test paper to test this internal control test sample, whether comprising an amplification internal control nucleotide sequence (i.e. this internal control nucleotide sequence of amplification in this first stage Polymerase Chain Reaction) in this first stage response sample to check, this amplification internal control nucleotide sequence presence or absence can reflect that whether this first stage Polymerase Chain Reaction is successful。During enforcement, taken out 45 microlitres as this internal control test sample by this first stage response sample obtained in step S30, this internal control test paper is steeped into this internal control test sample to test。
In the present embodiment, the structure of this internal control test paper is as shown in Figure 2, this internal control test paper 1 comprises a supporter 10, one corpse or other object for laboratory examination and chemical testing layer 12, one complex releasing layer 14, one inspected layer 16 and an absorbed layer 18, this supporter 10 be arranged at this internal control test paper 1 bottom and as its basis, this corpse or other object for laboratory examination and chemical testing layer 12, this complex releasing layer 14, this inspected layer 16 and this absorbed layer 18 are sequentially arranged by the front end (figure left) (figure right) to the back-end of this supporter 10, wherein the rear end of this corpse or other object for laboratory examination and chemical testing layer 12 is covered in the front end of this complex releasing layer 14, the rear end of this complex releasing layer 14 is covered in the front end of this inspected layer 16, the front end of this absorbed layer 18 is then covered in the rear end of this inspected layer 16, wherein, containing a labeled complex in this complex releasing layer 14, this inspected layer 16 comprises inspection line 160 and a control line 162。
The internal control test paper used in the present embodiment utilizes immunity sandwich to detect, this inspection line 160 comprises an antibody, this antibody can specifically bind to this antigen that this amplification internal control nucleotide sequence connects, this labeled complex comprises a mark substance and a receptor, this mark substance such as gold colloidal or other equivalent effect thing can colour generation to reflect the existence of this labeled complex, this receptor then also can specifically bind to this part that this amplification internal control nucleotide sequence connects, as previously mentioned, in the present embodiment, 5 ' ends of one DNA (deoxyribonucleic acid) of this amplification internal control nucleotide sequence carry out labelling using Digitoxin as this antigen, 5 ' ends of another burst of DNA (deoxyribonucleic acid) then carry out labelling with biotin as this part, therefore the present embodiment can resist the antibody (anti-DIGantibody) of Digitoxin as this antibody with in conjunction with this antigen Digitoxin of labelling on this amplification internal control nucleotide sequence, and using avidin (avidin) as this receptor with in conjunction with this part biotin of labelling on this amplification internal control nucleotide sequence。
This control line 162 then comprises the material can being combined with this labeled complex, as being in particular this receptor that can be combined in this mark substance and the part designed, such as fill this part protein bound with one, in the present embodiment, this control line 162 comprises and bovine serum albumin (bovineserumalbumin, BSA) biotin combined, bovine serum albumin is as this filling albumen, non-specific combination can be reduced, biotin is then as this part, and avidin as this receptor can be combined with colour generation in this labeled complex。
After this sample layer 12 of this internal control test paper 1 front end is immersed this internal control test sample, flow in the rear end that this internal control test sample is affected to this internal control test paper 1 by capillary force, when this complex releasing layer 14, this internal control test sample will contact with this labeled complex, if comprising this amplification internal control nucleotide sequence in this internal control test sample, this labeled complex would be incorporated into this amplification internal control nucleotide sequence, when arriving this p-wire 160, this amplification internal control nucleotide sequence being combined with this labeled complex would be incorporated into this antibody and makes this p-wire 160 colour generation, this labeled complex not being combined with this amplification internal control nucleotide sequence then advances to this control line 162, be combined with this part and make this control line 162 colour generation。
If this unsuccessful amplification of internal control nucleotide sequence, when then this internal control test sample arrives this p-wire 160, this amplification internal control nucleotide sequence (and being incorporated into this labeled complex of this amplification internal control nucleotide sequence) will not be had to be captured by this antibodies, this p-wire 160 will not colour generation, and this control line 162 still can colour generation。
Separately, if this non-colour generation of control line 162, then it represents that manufacture or the use procedure of this internal control test paper 1 have unusual condition, and the test result of this internal control test paper 1 is invalid, should retest this internal control test sample with another internal control test paper。
As shown in Figure 3A, this p-wire 160 and this control line 162 all colour generation of this internal control test paper 1, it is shown as internal control positive findings, expression can record this amplification internal control nucleotide sequence (i.e. this internal control nucleotide sequence Successful amplification)；As shown in Figure 3 B, this non-colour generation of p-wire 160 of this internal control test paper 1, and this control line 162 colour generation, it is shown as internal control negative findings, represents this unsuccessful amplification of internal control nucleotide sequence；As shown in Fig. 3 C and Fig. 3 D, all non-colour generation of this control line 162 of this internal control test paper 1, it is shown as fail result, this internal control test paper 1 represented in Fig. 3 C and Fig. 3 D has exception, and test result is invalid。
If judging to record this amplification internal control nucleotide sequence (this internal control nucleotide sequence not Successful amplification in step S40) in step S50, represent and this nucleic acid samples (with this first stage response sample) is likely to containing Polymerase Chain Reaction inhibiting substances, then carry out step S62, this first stage response sample does not go on this second stage Polymerase Chain Reaction, the material that Polymerase Chain Reaction inhibiting substances etc. can affect Polymerase Chain Reaction effect is avoided to cause the result of under-referral, step S10 should be re-started, again this nucleic acid samples is processed to remove the material affecting Polymerase Chain Reaction effect, even re-fetch Sputum samples and by Sputum samples re-fetches this nucleic acid samples。
If judging to record this amplification internal control nucleotide sequence (this internal control nucleotide sequence Successful amplification in step S40) in step S50, representing in this nucleic acid samples (with this first stage response sample) should (or the content of Polymerase Chain Reaction inhibiting substances be low without Polymerase Chain Reaction inhibiting substances, Polymerase Chain Reaction will not be affected), step S60 can be carried out, by this second stage introduction to adding to this first stage response sample, with in follow-up this second stage Polymerase Chain Reaction carried out, using this first stage target gene nucleotide sequence of amplification in this first stage Polymerase Chain Reaction as template, expand this second stage target gene nucleotide sequence (in the present embodiment use SEQIDNO.6 and SEQIDNO.7 nucleotide sequence as this second stage introduction to)。
As previously mentioned, in the present embodiment, this second stage target gene nucleotide sequence is the specific sequence of mycobacterium tuberculosis, checks this second stage target gene nucleotide sequence can as judging whether testee infects the foundation of mycobacterium tuberculosis after this second stage Polymerase Chain Reaction。
In step S60, four kinds of deoxyribose triphosphoric acids of the mixing that 0.2 microlitre deoxyribonucleic acid polymerase, 44.8 microliters polymerase reaction buffers and reaction density are each 0.2 micro-mole concentration should be added further, to carry out this second stage Polymerase Chain Reaction in step S70 in PCR (platoon) microcentrifugal tube。
In step S70, carrying out this second stage Polymerase Chain Reaction to expand this second stage target gene nucleotide sequence, in the present embodiment, in this second stage Polymerase Chain Reaction, reaction condition is as follows:
After step S70, can will can assemble the specific dna probe with specificity concretion mycobacterium nucleic acid sequence and bring out the enzyme of color reaction and added this second stage response sample by matter further, and the cold light readings of this second stage response sample is read with cold light instrument, this second stage target gene nucleotide sequence (can by the identification of above-mentioned specific dna probe institute) whether is contained in this second stage response sample to check, in the present embodiment, with 10 seconds cold light readings more than 100, 000 relative light unit (relativelightunit, RLU person) for mycobacterium tuberculosis positive findings, within 10 seconds, cold light readings is less than 25, 000 relative light unit is mycobacterium tuberculosis negative findings, if cold light readings was between 25 in 10 seconds, 000 to 100, 000 relative light unit then must re-start inspection by step S20。
Referring to Fig. 4 and Fig. 5, it is the internal control test paper test result figure and cold light readings result figure of second embodiment of the invention；In the present embodiment, to comprise the positive control group of M. tuberculosis genes body and the feminine gender control group without M. tuberculosis genes body, it is separately added into or is added without polymerase inhibitors matter (citrate, citric acid or ethylenediaminetetraacetic acid), and carry out the step such as first embodiment。
As shown in Figure 4, in positive control group and negative control group, add any polymerase inhibitors matter person, the test result of internal control test paper is all internal control feminine gender, again as shown in Figure 5, add the positive control group of any polymerase inhibitors matter, the result of cold light readings is equally low with negative control group, display polymerase inhibitors matter can cause the result (positive control group cannot be detected cold light) of under-referral, and the situation containing polymerase inhibitors matter in a corpse or other object for laboratory examination and chemical testing can be detected by internal control test paper after first stage Polymerase Chain Reaction。
In sum, the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex of the present invention can expand first stage target gene nucleotide sequence and internal control nucleotide sequence in first stage Polymerase Chain Reaction simultaneously, and after first stage Polymerase Chain Reaction, confirm internal control nucleotide sequence whether Successful amplification, therefore screening can be done sth. in advance before second stage Polymerase Chain Reaction and be likely to result in the sample of under-referral result containing Polymerase Chain Reaction inhibiting substances, and avoid carrying out non-essential second stage Polymerase Chain Reaction。
Above is only presently preferred embodiments of the present invention, not it is used for limiting scope of the invention process, all equalizations done according to the shape described in the claims in the present invention scope, structure, feature and spirit change and modify, and all should be included in scope of the presently claimed invention。
<110>Asiagen Corp.
<120>quick-acting internal controls are built on the method in two-stage polymerisation enzyme chain reflex
<160>7
<210>1
<211>123
<212>DNA
<213>Flavivirusdenguevirustype4
<400>1
GCTTGAGCAAACCGTGCTGCCTGTAGCTCCGCCAACAACGGGAGGCGTAAAAATCCCGGGGAGGCCATGCGCCACGGAAGCTGTACGCGTGGCATATTGGACTAGCGGTTAGAGGAGACCCCT
<210>2
<211>20
<212>DNA
<213>mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>2
CGTGAGGGCATCGAGGTGGC
<210>3
<211>20
<212>DNA
<213>mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>3
GCGTAGGCGTCGGTCACAAA
<210>4
<211>12
<212>DNA
<213>Flavivirusdenguevirustype4
<400>4
GAGCAAACCGTGCTGCCTGTAG
<210>5
<211>13
<212>DNA
<213>Flavivirusdenguevirustype4
<400>5
AGGGGTCTCCTCTAACCGCTAGT
<210>6
<211>18
<212>DNA
<213>mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>6
AGATGCACCGTCGAACGG
<210>7
<211>19
<212>DNA
<213>mycobacterium tuberculosis (Mycobacteriumtuberculosis)
<400>7
GCCACGTAGGCGAACCCTG
Claims (10)
1. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex, it is characterised in that it comprises:
Obtain a nucleic acid samples；
By an internal control nucleotide sequence, a first stage introduction to and an internal control nucleic acid introduction to adding to this nucleic acid samples, carry out a first stage Polymerase Chain Reaction, generate a first stage response sample；
Taken out part as an internal control test sample by this first stage response sample, and utilize an internal control test paper to test this internal control test sample to check whether this first stage response sample comprises an amplification internal control nucleotide sequence；And
If this internal control test paper records this amplification internal control nucleotide sequence, by a second stage introduction to adding to this first stage response sample, carry out a second stage Polymerase Chain Reaction, generate a second stage response sample。
2. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex as claimed in claim 1, it is characterized in that, wherein expanding a first stage target gene nucleotide sequence in this first stage introduction is for this first stage Polymerase Chain Reaction, this first stage response sample comprises an amplification first stage target gene nucleotide sequence。
3. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex as claimed in claim 2, it is characterized in that, wherein expanding a second stage target gene nucleotide sequence in this second stage introduction is for this second stage Polymerase Chain Reaction, this second stage response sample comprises an amplification second stage target gene nucleotide sequence。
4. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex as claimed in claim 1, it is characterized in that, wherein this internal control test paper comprises a p-wire, and this p-wire comprises an antibody, and this antibody is to specifically bind to the antigen that this amplification internal control nucleotide sequence connects。
5. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex as claimed in claim 4, it is characterised in that wherein this antigen is Digitoxin (digoxigenin), and this antibody is the antibody of primary antibodie Digitoxin。
6. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex as claimed in claim 1, it is characterized in that, wherein this internal control test paper comprises a labeled complex, this labeled complex be a receptor in conjunction with a mark substance, this receptor is to specifically bind to the part (ligand) that this amplification internal control nucleotide sequence connects。
7. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex as claimed in claim 6, it is characterised in that wherein this part is biotin (biotin), and this receptor is avidin (avidin)。
8. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex as claimed in claim 6, it is characterised in that wherein this mark substance is colloid gold。
9. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex as claimed in claim 6, it is characterised in that wherein this internal control test paper comprises a control line, and this control line comprises this part。
10. the method quick-acting internal controls built in two-stage polymerisation enzyme chain reflex as claimed in claim 6, it is characterized in that, wherein this internal control test paper comprises a control line, and this control line comprises a filling albumen, and this filling albumen not easily produces non-specific combination with this receptor。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410692880.3A CN105695561A (en) | 2014-11-26 | 2014-11-26 | Method for establishing quick acting internal control in two-stage polymerase chain reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410692880.3A CN105695561A (en) | 2014-11-26 | 2014-11-26 | Method for establishing quick acting internal control in two-stage polymerase chain reaction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105695561A true CN105695561A (en) | 2016-06-22 |
Family
ID=56294204
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410692880.3A Pending CN105695561A (en) | 2014-11-26 | 2014-11-26 | Method for establishing quick acting internal control in two-stage polymerase chain reaction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105695561A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307355A (en) * | 2007-11-30 | 2008-11-19 | 亚洲基因科技股份有限公司 | Test paper and method for checking mycobacteria tuberculosis and non-mycobacteria tuberculosis nucleic acid amplifying products |
| CN101957373A (en) * | 2010-08-20 | 2011-01-26 | 华东医学生物技术研究所 | Method for semi-quantitatively detecting pathogenic nucleic acid by adding internal control nucleic acid |
-
2014
- 2014-11-26 CN CN201410692880.3A patent/CN105695561A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307355A (en) * | 2007-11-30 | 2008-11-19 | 亚洲基因科技股份有限公司 | Test paper and method for checking mycobacteria tuberculosis and non-mycobacteria tuberculosis nucleic acid amplifying products |
| CN101957373A (en) * | 2010-08-20 | 2011-01-26 | 华东医学生物技术研究所 | Method for semi-quantitatively detecting pathogenic nucleic acid by adding internal control nucleic acid |
Non-Patent Citations (1)
| Title |
|---|
| ANKE DINKEL ET AL.: "Detection of Echinococcus multilocularis in the definitive host:coprodiagnosis by PCR as an alternative to necropsy", 《JOURNAL OF CLINICL MICROBIOLOGY》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zeng et al. | Rapid, ultrasensitive and highly specific biosensor for the diagnosis of SARS-CoV-2 in clinical blood samples | |
| Green et al. | What tests could potentially be used for the screening, diagnosis and monitoring of COVID-19 and what are their advantages and disadvantages | |
| WO2017181339A1 (en) | Method and kit for simultaneous detection of protein ligand and gene | |
| Boyles et al. | Incremental yield and cost of urine Determine TB-LAM and sputum induction in seriously ill adults with HIV | |
| Quinn | Recent advances in diagnosis of sexually transmitted diseases | |
| CN103698511B (en) | The purposes of Mycobacterium tuberculosis albumen in the product of preparation diagnosis latent tuberculosis infection | |
| Gaydos et al. | A narrative review of where we are with point-of-care sexually transmitted infection testing in the United States | |
| KR20130138775A (en) | Reagent composition for nucleic acid chromatography or immunochromatography, method for measurement by nucleic acid chromatography or immunochromatography, and kit for measurement by nucleic acid chromatography or immunochromatography | |
| US20210115496A1 (en) | Methods and Devices for Real-Time Diagnostic Testing (RDT) for Ebola and other Infectious Diseases | |
| Fernandes et al. | Recent advances in point of care testing for COVID-19 detection | |
| Chen et al. | A rapid, self-confirming assay for HIV: simultaneous detection of anti-HIV antibodies and viral RNA | |
| Lyu et al. | Automatic label-free immunoassay with high sensitivity for rapid detection of SARS-CoV-2 nucleocapsid protein based on chemiluminescent magnetic beads | |
| Bradbury et al. | On-demand nanozyme signal enhancement at the push of a button for the improved detection of SARS-CoV-2 nucleocapsid protein in serum | |
| Drain et al. | Duration of viral infectiousness and correlation with symptoms and diagnostic testing in non-hospitalized adults during acute SARS-CoV-2 infection: A longitudinal cohort study | |
| Rahimkhoei et al. | Nano‐based methods for novel coronavirus 2019 (2019‐nCoV) diagnosis: A review | |
| CN104561375A (en) | Isothermal amplification detection kit and detection method of new bunyavirus | |
| CN105695561A (en) | Method for establishing quick acting internal control in two-stage polymerase chain reaction | |
| JP2019508684A (en) | Methods and kits for the diagnosis of active tuberculosis | |
| CN109402127A (en) | One group of high affinity nucleic acid aptamers and its application specifically bound with Connective Tissue Growth Factor | |
| Schachter et al. | Discrepant analysis and screening for Chlamydia trachomatis | |
| CN103675293B (en) | The purposes of Rv3872, Rv0164 and/or Rv1926c albumen in the phthisical product of preparation diagnostic activities | |
| Czibere et al. | Verification of lateral flow antigen tests for SARS-CoV-2 by qPCR directly from the test device | |
| CN105256058B (en) | A kind of constant-temperature amplification detection kit and detection method of chikungunya fever virus | |
| EP1933148A1 (en) | Urinary immunochromatographic multiparameter detection cup | |
| Riegler et al. | Point-of-Care Testing for Sexually Transmitted Infections |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160622 |
|
| WD01 | Invention patent application deemed withdrawn after publication |