CN103305499A - Direct amplification reagent and its application - Google Patents
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Abstract
The invention discloses a PCR (polymerase chain reaction) amplification reagent and its application. The direct amplification reagent provided by the invention is the following 1) or 2): 1) the direct amplification reagent as shown includes a nonionic surfactant and an alcohol compound; and 2) the direct amplification reagent as shown includes nonionic surface active agents. Experiments involved in the invention prove that, the direct amplification reagent provided in the invention can pass over the DNA extraction and purification steps to carry out PCR amplification or STR fluorescent multiplex amplification directly on an FTA blood card, blood and other common biological samples so as to obtain an ideal result.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of direct amplifing reagent and application thereof.
Background technology
Polymerase chain reaction (Polymerase Chain reaction), being called for short PCR is a kind of important Protocols in Molecular Biology.From late 1980s, the people such as Mullis have developed since the round pcr, and this technology has been widely used in each association area such as biology, medical science, medical jurisprudence based on its distinctive technical superiority.
The quality and quantity of template DNA is directly determining the success or failure of PCR reaction usually.Therefore, the method for inspection of a large amount of PCR-based technology and detection means need to be carried out DNA extraction and the purifying of biological specimen before amplification.Verify as example with forensic dna, its main test flow process comprise DNA extraction, DNA quantitatively, four steps such as pcr amplification, product detection.Yet conventional DNA extraction method such as organic method (phenol chloroform method), silica bead method, Chelex method etc. are usually directed to following technological deficiency: 1) complex operation step; 2) length consuming time; 3) expend a large amount of human and material resources, financial resources; 4) extraction efficiency is low; 5) relate to the use of poisonous and harmful organic reagent; 6) can't realize extracting automatization etc.Magnetic bead DNA extraction method can Effective Raise DNA extraction efficient, and the step that simplifies the operation avoids using noxious solvent, and can realize that the automatization of high pass DNA extracts.But when in the face of ultra-large biological specimen high-throughput check, the method still exists many weak points, 1) although can realize automatization, need to rely on large automatic operation element station, hardware has high input; 2) easily cause crossed contamination between sample; 3) mostly commercialization of reagent, reagent cost has high input; 4) supporting consumptive material consumption is large; 5) although the operating time that this method reduces greatly, full operation still needs to consume 1.5-3 hour;
In order to capture the bottleneck of these sample high-throughput checks, improve the checkability of sample, a lot of researchists attempt to save the DNA extraction step, directly carry out pcr amplification operation, i.e. so-called direct amplification technique (Direct PCR) for an amount of biological specimen.
Direct PCR (Direct PCR) claims again direct amplification technique or the hands-free amplification technique of getting, it can directly add original sample without DNA extraction and other pre-treatment step the PCR reaction system and obtain amplified production, reduce checking procedure, reduce inspection cost, the a kind of of round pcr, for biological specimen is rapidly and efficiently checked, this will be a huge revolutionary advancement undoubtedly.Yet, find in the actual application, adopt traditional direct amplifing reagent can not obtain desirable experimental result.Its chief reason is that biological specimen usually is accompanied by a large amount of PCR inhibitor and is released in the PCR system, and dna polymerase activity is reduced or inactivation, thereby seriously reduces PCR efficient even cause the generation of negative findings.
Along with the fast development of this technology, the researchist sets up the direct amplifing reagent system of the multiple difference of polysaccharase that has anti-inhibition ability by adding different PCR tougheners or use in recent years.A lot of direct amplifing reagents of commercialization also successively are pushed out and are applied to practice.
The biological specimen such as blood, saliva is the common sample type that directly increases, and these samples are usually located on the different carriers.Common bearer type has cotton fibre, filter paper, 903 cards, also has in addition FTA card, FTA oral cavity card (indicator type) etc.The FTA card be a kind of for various biological samples collect, transport, file and its nucleic acid of purifying are gone forward side by side performing PCR, SNP, RT-PCR, RFLP and Restriction Endonuclease effect etc. are analyzed and the common carrier of design, have wide range of applications.The FTA card is through the chemical formulation dipping of patent, and energy dissolved cell film also changes protein properties, and nucleic acid is fixed and can protects its erosion that avoids UV phototoxis and microorganism and fungi, is beneficial to the preservation of DNA.Current, the FTA card is widely used in DNA check field by numerous a forensic DNA laboratories as the carrier of preserving sample.Simultaneously, it should be noted that FTA card preservation sample (FTA blood card, oral cavity card) is in the process of carrying out the DNA check, owing to containing a large amount of PCR inhibitor, need to extract or wash processing, otherwise the phenomenon or increase unsuccessfully of very easily causing not exclusively increasing.
Further quickening along with China DNA database establishment paces, be badly in need of improving Sample speed, the Direct PCR technology is one of effective way that addresses this problem, and current all commercializations are directly expanded reagent the FTA card is directly increased all based on certain system scope (20-25uL) or the corresponding low punching size (0.5-0.75) that reduces the FTA card.When adopting low system (8ul-10ul) to carry out conventional big or small FTA blood card or the amplification of oral cavity card, show obvious inhibition or fall flat, have two and significantly use bottlenecks: 1) can not effectively save inspection cost; 2) owing to using low system to cause the appearance that reduces to cause new series of problems in aperture, more be subjected to the impact of electrostatic force large such as less blood card (0.5mm), easily cause crossed contamination, 0.5mm the aperture can only adopt the method for Manual punching, existing BSD automatic punching device can not reach the service requirements of 0.5mm, the flow process that can not effectively simplify the operation, and be unfavorable for the high-throughput check of sample.
Summary of the invention
The invention provides a kind of direct amplifing reagent.
Direct amplifing reagent provided by the invention is following 1) or 2):
1) the direct amplifing reagent shown in comprises nonionic surface active agent and alcohol compound;
2) the direct amplifing reagent shown in comprises nonionic surface active agent;
Described nonionic surface active agent be Tween 20, Tween60, Tween80, Tween65, Tween85, TritonX-100, Nonidet P40 (NP40), octyl phenyl-macrogol (
CA-630), at least a in polyoxyl 10 oleyl ether and the N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE;
Described alcohol compound is at least a in PEG200, PEG300, PEG400, PEG600, PEG6000, PEG8000, ethylene glycol, glycerol, propylene glycol and the hexylene glycol.
In the above-mentioned direct amplifing reagent, 1) the direct amplifing reagent shown in is following 1a) or 1b):
Described 1a) in, described nonionic surface active agent is N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE and octyl phenyl-macrogol, and described alcohol compound is PEG6000;
Described 1b) in, described nonionic surface active agent is Tween 20, and described alcohol compound is PEG6000;
Described 2) in, described nonionic surface active agent is Tween 20 and octyl phenyl-macrogol.
In the above-mentioned direct amplifing reagent,
Described 1a) in, the proportioning of described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE, described octyl phenyl-macrogol and described PEG6000 is (0.12-0.3) ml: (0.12-0.3) ml: 5g; The proportioning of described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE, described octyl phenyl-macrogol and described PEG6000 further is specially 0.2ml: 0.2ml: 5g;
Described 1b) in, the proportioning of described Tween 20 and described PEG6000 is (0.8-1) ml: 5g; The proportioning of described Tween20 and described PEG6000 further is specially 0.9ml: 5g;
Described 2) in, the volume ratio of described Tween 20 and described octyl phenyl-macrogol is (0.8-1): 0.12; The volume ratio of described Tween 20 and described octyl phenyl-macrogol further is specially 0.9: 0.12.
In the above-mentioned direct amplifing reagent,
Described 1a), 1b) or 2) in comprise also that all pH is that 8.3 concentration are Tris-HCl damping fluid, the MgCl of 20mmol/L
2, dNTPs, KCl, NH
4Cl, BSA, archaeal dna polymerase and water;
Described 1a) be that 8.3 concentration are 20mmol/L Tris-HCl damping fluid, described MgCl by described pH
2, described dNTPs, described KCl, described NH
4Cl, described BSA, described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE, described octyl phenyl-macrogol, described PEG6000, archaeal dna polymerase and described water form;
Described 1b) be that 8.3 concentration are 20mmol/L Tris-HCl damping fluid, described MgCl by described pH
2, described dNTPs, described KCl, described NH
4Cl, described BSA, described Tween 20, described PEG6000, described archaeal dna polymerase and described water form;
Described 2) be that 8.3 concentration are 20mmol/L Tris-HCl damping fluid, described MgCl by described pH
2, described dNTPs, described KCl, described NH
4C
l, described BSA, described Tween 20, described octyl phenyl-macrogol, described NA polysaccharase and described water forms.
In the above-mentioned direct amplifing reagent, described 1a) in, described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE is at described 1a) in concentration be 0.12%-0.3% (volumn concentration); Described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE is at described 1a) in concentration further be specially 0.2% (volumn concentration);
Described octyl phenyl-macrogol is at described 1a) in concentration be 0.12%-0.3% (volumn concentration); Described octyl phenyl-macrogol is at described 1a) in concentration further be specially 0.2% (volumn concentration);
Described PEG6000 is at described 1a) in concentration be 4.5%-5.5% (quality percentage composition); Described PEG6000 is at described 1a) in concentration further be specially 5% (quality percentage composition);
Described 1b) in, described Tween 20 is at described 1b) in concentration be 0.8%-1% (volumn concentration), described Tween 20 is at described 1b) in concentration further be specially 0.9% (volumn concentration);
Described PEG6000 is at described 1b) in concentration be 4.5%-5.5% (quality percentage composition); Described PEG6000 is at described 1b) in concentration further be specially 5% (quality percentage composition);
Described 2) in, described Tween 20 is described 2) in concentration be 0.8%-1% (volumn concentration); Described Tween 20 is described 2) in concentration be specially 0.9% (volumn concentration);
Described octyl phenyl-macrogol is described 2) in concentration be 0.1%-0.2% (volumn concentration); Described octyl phenyl-macrogol is described 2) in concentration further be specially 0.12% (volumn concentration).
At described 1a), 1b) and 2) in, described MgCl
2Concentration in the described direct amplifing reagent of its correspondence is 1.6mmol/L; The concentration of each described dNTP in the described direct amplifing reagent of its correspondence is 200uM; The concentration of described KCl in the described direct amplifing reagent of its correspondence is 50mmol/L; Described NH
4The concentration of Cl in the described direct amplifing reagent of its correspondence is 25mmol/L; The concentration of described BSA in the described direct amplifing reagent of its correspondence is 700ug/ml-1200ug/ml; The concentration of described BSA in the described direct amplifing reagent of its correspondence specifically further is 980ug/ml or 1200ug/ml; The concentration of described archaeal dna polymerase in the described direct amplifing reagent of its correspondence is 0.1U/ul-0.4U/ul, and the concentration of described archaeal dna polymerase in the described direct amplifing reagent of its correspondence further is specially 0.3U/ul or 0.4U/ul.
Another object of the present invention provides a kind of direct amplification kit.
Direct amplification kit provided by the invention comprises above-mentioned direct amplifing reagent.
The application in the product that directly increases for the preparation of sample to be tested of above-mentioned direct amplifing reagent or above-mentioned test kit also is the scope of protection of the invention.
In above-mentioned application, described sample to be tested is that the aperture is that the FTA blood card of 1.2mm, FTA oral cavity card or the aperture that the aperture is 1.2mm are the common filter paper blood of 1.2mm card;
The amplification system volume of described direct amplification is 8ul-10ul, and the amplification system volume of described direct amplification further is specially 8ul or 10ul;
Described direct amplifing reagent is the STR fluorescent composite amplification.
The 3rd purpose of the present invention provides a kind of method for the direct amplifing reagent of sample to be tested.
Method provided by the invention comprises the steps: to carry out direct amplifing reagent with above-mentioned direct amplifing reagent or above-mentioned test kit and primer pair sample to be tested, obtains amplified production.
In the aforesaid method, described sample to be tested is that the aperture is that the FTA blood card of 1.2mm, FTA oral cavity card or the aperture that the aperture is 1.2mm are the common filter paper blood of 1.2mm card;
The amplification system volume of described direct amplification is 8ul-10ul, and the amplification system volume of described direct amplification further is specially 8ul or 10ul;
Described direct amplification is the STR fluorescent composite amplification.
Primer in the aforesaid method is the primer in the DNATyper15 test kit (available from Material Evidence Identification Center, Ministry of Public Security).
Of the present invention experimental results show that, direct amplifing reagent provided by the invention, can cross DNA extraction and purification step directly to FTA blood card, the common biological specimen such as blood carries out pcr amplification or STR fluorescent composite amplification, obtain desired result, can be under extremely low amplifing reagent system (8-10 μ l), realization is to the direct amplification of 1.2mm FTA blood card and oral cavity card, obtain high-quality STR somatotype collection of illustrative plates, single is detected as power and reaches that (all are verified the sample number that can obtain complete STR information in the sample is X more than 93%, total sample number is Y, above-mentioned check success ratio is X/Y), therefore reagent provided by the invention can satisfy the requirement that reduces cost and can effectively break through above-mentioned many technical bottlenecks again, realizes high-level efficiency, the high success rate amplification.
Description of drawings
Fig. 1 is standard STR somatotype collection of illustrative plates
Fig. 2 is the direct amplification that 1.2mm FTA blood is stuck in 8 μ l reagent systems
Fig. 3 is the direct amplification that 1.2mm FTA blood is stuck in 10 μ l reagent systems
Fig. 4 is the direct amplification that 1.2mm FTA oral cavity is stuck in 10 μ l reagent systems
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Concentration among the following embodiment is the system final concentration if no special instructions.
Material used among the following embodiment is exemplified below:
Nonionic surface active agent is Tween 20, Tween60, Tween80, Tween65, Tween85, TritonX-100, NP40, octyl phenyl-macrogol, polyoxyl 10 oleyl ether or N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE;
Alcohol compound is PEG200, PEG300, PEG400, PEG600, PEG6000, PEG8000, ethylene glycol, glycerol, propylene glycol or hexylene glycol;
FTA blood card sample, by the blood sample that Material Evidence Identification Center, Ministry of Public Security provides, the Whatman company of sale company of blood card, catalog number WB129237; Be cut into the blood card of different sizes by different needs.
The combination of primers of DNAtyper15 test kit is available from the primer in the DNAtyper15 test kit of Material Evidence Identification Center, Ministry of Public Security.
The pH value is that 8.3 (under the room temperature condition) 20mmol Tris-HCl damping fluid is available from U.S. Amresco company, catalog number catalog number (Cat.No.): 0780C171; The water-soluble rear final concentration of Tris alkali is 20mmol, regulates the pH value to 8.0-8.3 with concentrated hydrochloric acid);
BSA (U.S. Amresco company, catalog number E588);
Octyl phenyl-macrogol (IPEGAL CA630, U.S. Sigma company, catalog number I8896);
TritonX-100 (U.S. Sigma company, catalog number T8787);
Taq archaeal dna polymerase (Roche company, catalog number 12032945001);
Tween20 (Amresco company, catalog number 0630C404);
NP40 (Luo Shi, catalog number 11332473001);
PEG200 (U.S. sigma, catalog number p3015);
GoldTaq archaeal dna polymerase (Roche company, article No.: 12032945001);
PEG6000 (German Merck company, article No. 807491);
The acquisition of embodiment 1, standard STR somatotype collection of illustrative plates
Get the 3.0mm FTA blood card sample (blood sample that Material Evidence Identification Center, Ministry of Public Security provides, the Whatman company of sale company of blood card, catalog number WB129237), adopt the Chelex-100 method to carry out the DNA extraction operation according to Standard Operating Procedure, adopt DNATyper15 test kit (available from Material Evidence Identification Center, Ministry of Public Security) to carry out the STR fluorescent composite amplification according to operation instructions, carry out the electrophoretic separation fluoroscopic examination at ABI3130xl, warp
Result such as Fig. 1 that software analysis obtains.
Embodiment 2, directly preparation and the application thereof of amplifing reagent
One, 1.2mm FTA blood is stuck in the direct amplification (with direct amplifing reagent 1a) in the 8 μ l reagent systems
This part experiment mainly suppresses ability for the high resistance that further proves reagent of the present invention.
Method one:
1, the directly preparation of amplifing reagent 1a
Directly amplifing reagent 1a is by 20mmol Tris-HCl, 1.6mM MgCl
2, 200uM each dNTP, 50mM KCl, 25mM NH
4Cl; 1200ug/ml BSA, 0.2% (volumn concentration) octyl phenyl-macrogol, 0.2% (volumn concentration) N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE; 5% (quality percentage composition) PEG6000,0.3U/ μ l GoldTaq archaeal dna polymerase and water form.
2, the application of the direct amplifing reagent 1a of 8 μ l in 1.2mm FTA blood card directly increases
The FTA blood card (blood sample that Material Evidence Identification Center, Ministry of Public Security provides with 1.2mm, the Whatman company of sale company of blood card, catalog number WB129237) adds the direct amplifing reagent 1a of 8 μ l, primer adopts the combination of primers of DNAtyper15 test kit, carry out the STR fluorescent composite amplification, the system volume is 8ul.
STR fluorescent composite amplification condition is as follows: 75 ℃, 15min is hatched; 95 ℃, 11min sex change and cracking; 94 ℃, 30s, 59 ℃, 2min, 72 ℃, 1min, 28 circulations; 60 ℃, 30min, 25 ℃ of permanent thermal insulations.
STR fluorescent composite amplification result obtains standard STR somatotype trace analysis with embodiment 1 shown in Fig. 2 a, can find out, the STR somatotype collection of illustrative plates complete display that direct amplifing reagent 1a obtains, balance is good, and the artificial product of specificity occurs nothing but, loses or significantly suppress phenomenon without band to occur.
Adopt identical method, with 8ul commercial reagents (ABI company, catalog number (Cat.No.): 4467831, Products Show uses system to be 25ul) carry out controlled trial, the result can find out shown in Fig. 2 b, and excessive FTA blood hole clipping footpath has produced strong inhibition to reagent system, the large fragment Loss is serious, shows as common PCR and suppresses phenomenon.
Adopt identical method, with the direct amplifing reagent 1a of 25 μ l the FTA blood card of 1.2mm is carried out the STR fluorescent composite amplification, result and 8 μ l systems are without significant difference.
Above description of test, in being low to moderate the reagent system of 8ul, direct amplifing reagent 1a of the present invention still can directly increase for 1.2mmFTA blood card, can obtain complete, high quality STR somatotype collection of illustrative plates, and saves inspection cost.
Method two:
1, the directly preparation of amplifing reagent 1a: basic identical with method one; different is 0.12% (volumn concentration) octyl phenyl-macrogol; 0.12% (volumn concentration) N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE, 4.5% (quality percentage composition) PEG6000.
2, the application of the direct amplifing reagent 1a of 8 μ l in 1.2mm FTA blood card directly increases: method is identical with method one, and result and method one are without significantly difference.
Method three:
1, the directly preparation of amplifing reagent 1a: basic identical with method one; different is 0.3% (volumn concentration) octyl phenyl-macrogol; 0.3% (volumn concentration) N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE, 5.5% (quality percentage composition) PEG6000.
2, the application of the direct amplifing reagent 1a of 8 μ l in 1.2mm FTA blood card directly increases: method is identical with method one, and result and method one are without significantly difference.
Two, 1.2mm FTA blood is stuck in the direct amplification (with direct amplifing reagent 1b) in the 10 μ l reagent systems
Method one:
1, the directly preparation of amplifing reagent 1b
Directly amplifing reagent 1b is 8.3 (under the room temperature condition) 20mmol Tris-HCl by the pH value, 1.6mM MgCl
2, 200uM each dNTP, 50mM KCl, 25mM NH
4Cl, 1200ug/ml BSA, 0.9% (volumn concentration) Tween20,5% (quality percentage composition) PEG6000,0.4U/ μ l GoldTaq archaeal dna polymerase and water form.
2, the application of the direct amplifing reagent 1b of 10 μ l in 1.2mm FTA blood card directly increases
The FTA blood card of 1.2mm is added the direct amplifing reagent 1b of 10ul, and primer adopts the combination of primers of DNAtyper15 test kit, carries out the STR fluorescent composite amplification, and the system volume is 10ul.
STR fluorescent composite amplification condition is as follows: 75 ℃, 15min is hatched; 95 ℃, 11min sex change and cracking; 94 ℃, 30s, 59 ℃, 2min, 72 ℃, 1min, 28 circulations; 60 ℃, 30min, 25 ℃ of permanent thermal insulations.
STR fluorescent composite amplification result obtains standard STR somatotype trace analysis with embodiment 1 shown in Fig. 3 a, can find out, the STR somatotype collection of illustrative plates complete display that direct amplifing reagent 1b obtains, balance is good, and the artificial product of specificity occurs nothing but, loses or significantly suppress phenomenon without band to occur.
Adopt identical method, with 10ul commercial reagents (ABI company, catalog number (Cat.No.): 4467831, Products Show uses system to be 25ul) carry out controlled trial, the result can find out shown in Fig. 3 b, and excessive FTA blood hole clipping footpath has formed strong inhibition to reagent system, the large fragment Loss is serious, shows as common PCR and suppresses phenomenon.
Adopt identical method, with the direct amplifing reagent 1b of 25 μ l the FTA blood card of 1.2mm is carried out the STR fluorescent composite amplification, result and 10 μ l systems are without significant difference.
Above description of test, in the reagent system of 10ul, the direct amplifing reagent 1b of reagent of the present invention still can directly increase for 1.2mmFTA blood card, can obtain complete, high quality STR somatotype collection of illustrative plates, and saves inspection cost.
Method two:
1, the directly preparation of amplifing reagent 1b: basic identical with method one, that different is 0.8% (volumn concentration) Tween20,4.5% (quality percentage composition) PEG6000.
2, the application of the direct amplifing reagent 1b of 10 μ l in 1.2mm FTA blood card directly increases: method is identical with method one, and result and method one are without significantly difference.
Method three:
1, the directly preparation of amplifing reagent 1b: basic identical with method one, that different is 1% (volumn concentration) Tween20,5.5% (quality percentage composition) PEG6000.
2, the application of the direct amplifing reagent 1b of 10 μ l in 1.2mm FTA blood card directly increases: method is identical with method one, and result and method one are without significantly difference.
Three, 1.2mm FTA oral cavity is stuck in the direct amplification (with direct amplifing reagent 2) in the 10 μ l reagent systems
FTA oral cavity card also is class sample carriers commonly used, this part experiment main adaptability and anti-inhibition ability for further reagent.
Method one:
1, the directly preparation of amplifing reagent 2
Directly amplifing reagent 2 is 8.3 (under the room temperature condition) 20mmol Tris-HCl damping fluid, 1.6mM MgCl by the pH value
2, 200uM each dNTP, 50mM KCl, 25mM NH
4Cl, 980ug/ml BSA, 0.9% (volumn concentration) Tween20,0.12% (volumn concentration) octyl phenyl-macrogol, 0.4U/ μ l GoldTaq archaeal dna polymerase and water form.
2, the application of the direct amplifing reagent 2 of 10 μ l in 1.2mm FTA oral cavity card directly increases
(provide sample by Material Evidence Identification Center, Ministry of Public Security with the FTA oral cavity card of 1.2mm, the oral cavity card is available from Whatman company, catalog number WB129236) is added in the direct amplifing reagent 2 of 10 μ l, primer adopts the combination of primers of DNAtyper15 test kit, carry out the STR fluorescent composite amplification, the system volume is 10ul.
STR fluorescent composite amplification condition is as follows: 75 ℃, 20min is hatched; 95 ℃, 11min sex change and cracking; 94 ℃, 30s, 59 ℃, 2min, 72 ℃, 1min, 28 circulations; 60 ℃, 30min, 25 ℃ of permanent thermal insulations.
STR fluorescent composite amplification result obtains standard STR somatotype trace analysis with embodiment 1 shown in Fig. 4 a, can find out, the STR somatotype collection of illustrative plates complete display that direct amplifing reagent 2 obtains, balance is good, and the artificial product of specificity occurs nothing but, loses or significantly suppress phenomenon without band to occur.
Adopt identical method, with 10ul commercial reagents (ABI company, catalog number (Cat.No.): 4467831, Products Show uses system to be 25ul) carry out controlled trial, the result can find out shown in Fig. 4 b, for the oral cavity card, the result that 10ul reagent place of the present invention obtains is consistent with the result that 10ul commercialization reagent place obtains, and the collection of illustrative plates complete display reaches the service requirements of forensic identification.
Adopt identical method, carry out the STR fluorescent composite amplification with the FTA oral cavity card of 2 couples of 1.2mm of the direct amplifing reagent of 25 μ l, result and 10 μ l systems are without significant difference.
Method two:
1, the directly preparation of amplifing reagent 2: basic identical with method one, different is 0.8% (volumn concentration) Tween20,0.1% (volumn concentration) octyl phenyl-macrogol.
2, the application of the direct amplifing reagent 2 of 10 μ l in 1.2mm FTA oral cavity card directly increases: method is identical with method one, and result and method one are without significantly difference.
Method three:
1, the directly preparation of amplifing reagent 2: basic identical with method one, different is 1% (volumn concentration) Tween20,0.2% (volumn concentration) octyl phenyl-macrogol.
2, the application of the direct amplifing reagent 2 of 10 μ l in 1.2mm FTA oral cavity card directly increases: method is identical with method one, and result and method one are without significantly difference.
Claims (10)
1. a direct amplifing reagent is following 1) or 2):
1) the direct amplifing reagent shown in comprises nonionic surface active agent and alcohol compound;
2) the direct amplifing reagent shown in comprises nonionic surface active agent;
Described nonionic surface active agent is at least a in Tween 20, Tween60, Tween80, Tween65, Tween85, TritonX-100, Nonidet P40, octyl phenyl-macrogol, polyoxyl 10 oleyl ether and the N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE;
Described alcohol compound is at least a in PEG200, PEG300, PEG400, PEG600, PEG6000, PEG8000, ethylene glycol, glycerol, propylene glycol and the hexylene glycol.
2. direct amplifing reagent according to claim 1 is characterized in that:
1) the direct amplifing reagent shown in is following 1a) or 1b):
Described 1a) in, described nonionic surface active agent is N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE and octyl phenyl-macrogol, and described alcohol compound is PEG6000;
Described 1b) in, described nonionic surface active agent is Tween 20, and described alcohol compound is PEG6000;
Described 2) in, described nonionic surface active agent is Tween 20 and octyl phenyl-macrogol.
3. direct amplifing reagent according to claim 1 and 2 is characterized in that:
Described 1a) in, the proportioning of described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE, described octyl phenyl-macrogol and described PEG6000 is (0.12-0.3) ml: (0.12-0.3) ml: 5g; The proportioning of described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE, described octyl phenyl-macrogol and described PEG6000 further is specially 0.2ml: 0.2ml: 5g;
Described 1b) in, the proportioning of described Tween 20 and described PEG6000 is (0.8-1) ml: 5g; The proportioning of described Tween20 and described PEG6000 further is specially 0.9ml: 5g;
Described 2) in, the volume ratio of described Tween 20 and described octyl phenyl-macrogol is (0.8-1): 0.12; The volume ratio of described Tween 20 and described octyl phenyl-macrogol further is specially 0.9: 0.12.
4. arbitrary described direct amplifing reagent according to claim 1-3 is characterized in that:
Described 1a), 1b) or 2) in comprise also that all pH is that 8.3 concentration are Tris-HCl damping fluid, the MgCl of 20mmol/L
2, dNTPs, KCl, NH
4Cl, BSA, archaeal dna polymerase and water;
Described 1a) be that 8.3 concentration are 20mmol/L Tris-HCl damping fluid, described MgCl by described pH
2, described dNTPs, described KCl, described NH
4Cl, described BSA, described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE, described octyl phenyl-macrogol, described PEG6000, archaeal dna polymerase and described water form;
Described 1b) be that 8.3 concentration are 20mmol/L Tris-HCl damping fluid, described MgCl by described pH
2, described dNTPs, described KCl, described NH
4Cl, described BSA, described Tween 20, described PEG6000, described archaeal dna polymerase and described water form;
Described 2) be that 8.3 concentration are 20mmol/L Tris-HCl damping fluid, described MgCl by described pH
2, described dNTPs, described KCl, described NH
4Cl, described BSA, described Tween 20, described octyl phenyl-macrogol, described NA polysaccharase and described water form.
5. direct amplifing reagent according to claim 4 is characterized in that:
Described 1a) in, described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE is at described 1a) in concentration be 0.12%-0.3% (volumn concentration); Described N-capryloyl-N-METHYL-ALPHA-L-GLUCOSAMINE is at described 1a) in concentration further be specially 0.2% (volumn concentration);
Described octyl phenyl-macrogol is at described 1a) in concentration be 0.12%-0.3% (volumn concentration); Described octyl phenyl-macrogol is at described 1a) in concentration further be specially 0.2% (volumn concentration);
Described PEG6000 is at described 1a) in concentration be 4.5%-5.5% (quality percentage composition); Described PEG6000 is at described 1a) in concentration further be specially 5% (quality percentage composition);
Described 1b) in, described Tween 20 is at described 1b) in concentration be 0.8%-1% (volumn concentration), described Tween 20 is at described 1b) in concentration further be specially 0.9% (volumn concentration);
Described PEG6000 is at described 1b) in concentration be 4.5%-5.5% (quality percentage composition); Described PEG6000 is at described 1b) in concentration further be specially 5% (quality percentage composition);
Described 2) in, described Tween 20 is described 2) in concentration be 0.8%-1% (volumn concentration); Described Tween 20 is described 2) in concentration be specially 0.9% (volumn concentration);
Described octyl phenyl-macrogol is described 2) in concentration be 0.1%-0.2% (volumn concentration); Described octyl phenyl-macrogol is described 2) in concentration further be specially 0.12% (volumn concentration).
6. it is characterized in that according to claim 4 or 5 described direct amplifing reagents:
At described 1a), 1b) and 2) in, described MgCl
2Concentration in the described direct amplifing reagent of its correspondence is 1.6mmol/L; The concentration of each described dNTP in the described direct amplifing reagent of its correspondence is 200uM; The concentration of described KCl in the described direct amplifing reagent of its correspondence is 50mmol/L; Described NH
4The concentration of Cl in the described direct amplifing reagent of its correspondence is 25mmol/L; The concentration of described BSA in the described direct amplifing reagent of its correspondence is 700ug/ml-1200ug/ml; The concentration of described BSA in the described direct amplifing reagent of its correspondence specifically further is 980ug/ml or 1200ug/ml; The concentration of described archaeal dna polymerase in the described direct amplifing reagent of its correspondence is 0.1U/ul-0.4U/ul, and the concentration of described archaeal dna polymerase in the described direct amplifing reagent of its correspondence further is specially 0.3U/ul or 0.4U/ul.
7. a direct amplification kit comprises arbitrary described direct amplifing reagent among the claim 1-6.
8. arbitrary described direct amplifing reagent or the application of test kit claimed in claim 7 in the product that directly increases for the preparation of sample to be tested among the claim 1-6.
9. application according to claim 8 is characterized in that:
Described sample to be tested is that the aperture is that the FTA blood card of 1.2mm, FTA oral cavity card or the aperture that the aperture is 1.2mm are the common filter paper blood of 1.2mm card;
The amplification system volume of described direct amplification is 8u1-10ul, and the amplification system volume of described direct amplification further is specially 8ul or 10ul;
Described direct amplifing reagent is the STR fluorescent composite amplification.
10. one kind is used for the method that sample to be tested directly increases, and comprises the steps: directly to increase with arbitrary described direct amplifing reagent or test kit claimed in claim 7 and primer pair sample to be tested among the claim 1-6, obtains direct amplified production;
Described sample to be tested is specially the FTA blood card that the aperture is 1.2mm, FTA oral cavity card or the aperture that the aperture is 1.2mm is the common filter paper blood of 1.2mm card;
The amplification system volume of described direct amplification is specially 8ul-10ul, and the amplification system volume of described direct amplification further is specially 8ul or 10ul;
Described direct amplification is specially the STR fluorescent composite amplification.
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| CN108048547A (en) * | 2017-12-22 | 2018-05-18 | 美因健康科技（北京）有限公司 | The method that blood Direct PCR carries out Taqman partings |
| CN109295174A (en) * | 2018-10-22 | 2019-02-01 | 益善生物技术股份有限公司 | A kind of reagent set for detecting fungal infection, kit and detection method |
| JPWO2019074004A1 (en) * | 2017-10-10 | 2020-09-17 | 積水メディカル株式会社 | SNP detection method |
| CN111676216A (en) * | 2020-06-18 | 2020-09-18 | 合肥铼科生物科技有限公司 | Extraction-free inactivated virus sample nucleic acid preservation solution and preparation method and application thereof |
| CN112176038A (en) * | 2019-07-01 | 2021-01-05 | 申翌生物科技(杭州)有限公司 | Buffer composition |
| CN112481360A (en) * | 2020-12-17 | 2021-03-12 | 中国合格评定国家认可中心 | DNA hands-free direct amplification reagent for various case sample forensic science DNA typing detection |
| CN112921075A (en) * | 2021-03-31 | 2021-06-08 | 武汉友芝友医疗科技股份有限公司 | Hand-taking-free reagent for blood sample fluorescence PCR and application thereof |
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| CN103642919A (en) * | 2013-12-06 | 2014-03-19 | 亚能生物技术（深圳）有限公司 | Sample conditioning fluid based on denaturation precipitation as well as application thereof and nucleic acid releasing method |
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| CN108048547A (en) * | 2017-12-22 | 2018-05-18 | 美因健康科技（北京）有限公司 | The method that blood Direct PCR carries out Taqman partings |
| CN109295174A (en) * | 2018-10-22 | 2019-02-01 | 益善生物技术股份有限公司 | A kind of reagent set for detecting fungal infection, kit and detection method |
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| CN112481360A (en) * | 2020-12-17 | 2021-03-12 | 中国合格评定国家认可中心 | DNA hands-free direct amplification reagent for various case sample forensic science DNA typing detection |
| CN112481360B (en) * | 2020-12-17 | 2023-01-03 | 中国合格评定国家认可中心 | DNA hands-free direct amplification reagent for various case sample forensic science DNA typing detection |
| CN112921075A (en) * | 2021-03-31 | 2021-06-08 | 武汉友芝友医疗科技股份有限公司 | Hand-taking-free reagent for blood sample fluorescence PCR and application thereof |
| CN114480595A (en) * | 2022-01-30 | 2022-05-13 | 济凡生物科技（北京）有限公司 | PCR reaction solution and kit thereof for blood detection |
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