阅读说明：本技术 Lamp反应体系/分体系的分割处理和常温运输方法 (LAMP reaction system/split system segmentation treatment and normal temperature transportation method ) 是由 陈晓东 于 2020-12-31 设计创作，主要内容包括：本发明公开了一种LAMP反应体系/分体系的分割处理和常温运输方法,该方法以无菌无纺布/无菌无毛纸作为LAMP反应体系/分体系的无菌载体,首先将LAMP反应体系/分体系与海藻糖混合,得到混合液；再将无菌载体放入混合液中,充分浸润；再利用冷冻干燥机对上述的无菌载体进行冷冻干燥处理；最后将冷冻的无菌载体通过切割仪器进行等量切割,一管化分装,常温保存或运输。本发明将酶组分和PCR反应液组分分别固定在不同的无菌载体上,等量切割后按需求放入反应管内,重新组合成一管式LAMP反应体系,既避免了常规一管式混合冻干法假阳性率高的问题,又延长了LAMP反应体系的常温保存或运输期限。(The invention discloses a method for partitioning an LAMP reaction system/a split system and transporting the LAMP reaction system/the split system at normal temperature, which takes sterile non-woven fabrics/sterile non-woven paper as a sterile carrier of the LAMP reaction system/the split system, and firstly, the LAMP reaction system/the split system is mixed with trehalose to obtain a mixed solution; then putting the sterile carrier into the mixed solution for full infiltration; then, freeze-drying the sterile carrier by using a freeze dryer; and finally, equivalently cutting the frozen sterile carrier by a cutting instrument, subpackaging by one tube, and storing or transporting at normal temperature. According to the invention, the enzyme component and the PCR reaction solution component are respectively fixed on different sterile carriers, cut in equal amount and then placed into the reaction tube as required to be recombined into the one-tube LAMP reaction system, so that the problem of high false positive rate of the conventional one-tube mixed freeze-drying method is avoided, and the normal-temperature storage or transportation period of the LAMP reaction system is prolonged.)

1. A LAMP reaction system/split system segmentation treatment and normal temperature transportation method is characterized in that: the method comprises the following steps:

1) mixing the LAMP reaction system/sub-system with trehalose to obtain a mixed solution;

2) putting the sterile carrier into the mixed solution, and fully soaking; then, freeze-drying the sterile carrier by using a freeze dryer; wherein the sterile carrier is non-woven fabric/non-woolen paper;

3) and finally, equivalently cutting the frozen sterile carrier by a cutting instrument, subpackaging by one tube, and storing or transporting at normal temperature.

2. The LAMP reaction system/split system segmentation treatment and normal-temperature transportation method according to claim 1, characterized in that: in the step 1), the content of trehalose in each milliliter of mixed solution is 0-15 g.

3. The LAMP reaction system/split system segmentation processing and normal-temperature transportation method according to claim 2, characterized in that: when the mixed solution is obtained by mixing the LAMP reaction system and trehalose, the content of trehalose in each milliliter of the mixed solution is 10-15 g.

4. The LAMP reaction system/split system segmentation treatment and normal-temperature transportation method according to claim 3, characterized in that: when the LAMP reaction system is a DNA amplification system, the LAMP reaction system comprises primers, dNTPs, Bst DNA polymerase and reaction buffer solution for nucleic acid amplification;

or when the LAMP reaction system is an RNA amplification system, the LAMP reaction system comprises primers, dNTPs, Bst DNA polymerase, reaction buffer solution for nucleic acid amplification and reverse transcriptase/reverse transcriptase.

5. The LAMP reaction system/split system segmentation processing and normal-temperature transportation method according to claim 2, characterized in that: when the mixed solution is prepared by mixing the LAMP reaction system and trehalose,

if the LAMP reaction split system is an enzyme component system, the content of trehalose in each milliliter of mixed liquid is 10-15 g;

or if the LAMP reaction split system is a PCR reaction solution component system, the content of trehalose is 0-15 g; the PCR reaction solution component system comprises primers and dNTPs.

6. The LAMP reaction system/split system segmentation treatment and normal-temperature transportation method according to claim 5, characterized in that: the enzyme component system is a DNA enzyme component system and comprises Bst DNA polymerase and reaction buffer solution for nucleic acid amplification;

the enzyme component system is an RNA enzyme component system and comprises Bst DNA polymerase, dNTPs and reverse transcriptase or Bst DNA polymerase, reaction buffer solution for nucleic acid amplification and reverse transcriptase.

7. The LAMP reaction system/split system segmentation treatment and normal-temperature transportation method according to claim 4 or 6, characterized in that: the d NTPs are a mixture of d ATP, d TTP, d GTP and d CTP; the Bst DNA polymerase is a DNA polymerase having both 5 '→ 3' polymerase activity and strand displacement activity.

8. The LAMP reaction system/split system segmentation treatment and normal-temperature transportation method according to claim 1, characterized in that: in the step 2), the freeze drying time is 2 hours.

9. The method for using the sterile vector obtained by the LAMP reaction system/split system division treatment and normal-temperature transportation method according to claim 1, characterized by comprising the following steps:

a. adding water into a reaction tube containing a sterile carrier of the LAMP reaction system, and dissolving to obtain the LAMP reaction system for LAMP amplification reaction;

or b, adding water into a reaction tube of the sterile carrier containing a plurality of LAMP reaction subsystems, and dissolving to obtain the LAMP reaction system for LAMP amplification reaction.

Technical Field

The invention relates to the technical field of biochemical reagents, in particular to a method for partitioning an LAMP reaction system/split system and transporting the LAMP reaction system/split system at normal temperature.

Background

Notomi of Japan scholars in 2000 disclosed a novel isothermal Nucleic acid amplification technique suitable for gene diagnosis, namely, Loop-mediated isothermal amplification technique, which is called Loop-mediated isothermal amplification in English and is abbreviated as LAMP (Loop-mediated isothermal amplification) in journal of Nucleic Acids Res. The LAMP technology is characterized in that 4 specific primers are designed aiming at 6 regions of a target gene, under the action of strand displacement DNA Polymerase (Bst DNA Polymerase), amplification is carried out at the constant temperature of 60-65 ℃, and 10 can be realized within about 15-60 minutes⁹～10¹⁰Amplification of nucleic acids.

Compared with conventional PCR, the "LAMP" technology has the following advantages:

1. the sensitivity is high and is 2-5 orders of magnitude higher than that of the traditional nucleic acid amplification method;

2. the reaction time is short, thermal denaturation of the template is not needed, and the reaction can be completed within 30-60 minutes;

3. the whole reaction process is constant in temperature, no temperature circulation exists, and the clinical use does not depend on expensive professional detection equipment;

4. the reaction result can be judged by observing the generation of white turbidity or green fluorescence by naked eyes, electrophoresis and ultraviolet observation are not needed, the method is simple, convenient and quick, and ordinary personnel without professional skills can carry out quick diagnosis on the disease site.

In recent years, "LAMP" has been widely used in many countries for rapid detection of pathogenic microorganisms, such as SARS, influenza virus, Mycoplasma pneumoniae, Mycobacterium tuberculosis, and the like. However, in practical applications, the reagents required for the LAMP reaction: primers, d NTPs, polymerase, reaction buffer and the like all need to be stored for a long time at a low temperature of below 20 ℃ below zero and transported for a short time, and repeated freezing and thawing of reagents can influence the activity of the reagents and even cause failure. Meanwhile, various LAMP reaction reagent components (namely, primers, d NTPs, polymerase, reaction buffer solution and the like) except for the nucleic acid (DNA or RNA) of a sample to be detected are prepared into a tubular premix in advance, and false positive possibly occurs due to the influence of primer dimers on a system after long-term placement.

Therefore, the invention relates to a method for freeze-drying the primer and other LAMP reaction reagent components on different sterile carriers, which avoids the problem of high false positive rate of the conventional one-tube type mixed freeze-drying method, has important significance in the method of being convenient for normal-temperature storage or transportation, and has no related records at present.

Disclosure of Invention

At present, because the long-term placement of a system formed by mixing the components of a nucleic acid amplification reaction reagent into a tube type may cause the system to be influenced by primer dimers to cause false positives, and is not beneficial to long-term storage and transportation, the existing commercial kit generally packs enzyme components and PCR reaction liquid components into 2 different containers, the commercial kit needs to be stored for a long time and transported for a short time at a low temperature below-20 ℃, repeated freezing and thawing of the reagent can influence the activity of the reagent and even fails, and in the system freeze-dried powder prepared by the existing freeze-drying technology, the long-term placement of the system formed by mixing the primers and the enzyme components into a tube type may cause the system to be influenced by the primer dimers to cause false positives. The invention provides a method for dividing an LAMP reaction system/a split system and transporting the same at normal temperature, in particular to a method for dividing the LAMP reaction system/the split system and transporting the same at normal temperature except for nucleic acid (DNA or RNA) and water of a sample to be detected.

In order to achieve the purpose, the invention designs a LAMP reaction system/split system segmentation treatment and normal temperature transportation method, which comprises the following steps:

1) mixing the LAMP reaction system/sub-system with trehalose to obtain a mixed solution;

2) putting the sterile carrier into the mixed solution, and fully soaking; then, carrying out freeze drying treatment on the sterile carrier by using a freeze dryer (so that the LAMP reaction system/the split system is freeze-dried on the sterile carrier), wherein the sterile carrier is non-woven fabric/non-woven paper;

3) and finally, equally cutting the frozen sterile vector by a cutting instrument, subpackaging by one tube, and storing or transporting at normal temperature (the method realizes the segmentation treatment and normal temperature transportation of the LAMP reaction system/split system, and when the method is used, water can be added to restore the volume for LAMP amplification reaction).

Further, in the step 1), the content of trehalose in each milliliter of the mixed solution is 0-15 g.

And when the mixed solution is prepared by mixing the LAMP reaction system and trehalose, the content of trehalose in each milliliter of the mixed solution is 10-15 g.

Further, when the LAMP reaction system is a DNA amplification system, the LAMP reaction system comprises primers, dNTPs, Bst DNA polymerase and reaction buffer solution for nucleic acid amplification;

or when the LAMP reaction system is an RNA amplification system, the LAMP reaction system comprises primers, dNTPs, Bst DNA polymerase, reaction buffer solution for nucleic acid amplification and reverse transcriptase/reverse transcriptase.

Further, when the mixed solution is prepared by mixing the LAMP reaction system and trehalose,

if the LAMP reaction split system is an enzyme component system, the content of trehalose in each milliliter of mixed liquid is 10-15 g;

or if the LAMP reaction split system is a PCR reaction solution component system, the content of trehalose is 0-15 g; the PCR reaction solution component system comprises primers and dNTPs.

Still further, the enzyme component system is a DNA enzyme component system, and comprises Bst DNA polymerase and reaction buffer solution for nucleic acid amplification;

the enzyme component system is an RNA enzyme component system and comprises Bst DNA polymerase, dNTPs and reverse transcriptase or Bst DNA polymerase, reaction buffer solution for nucleic acid amplification and reverse transcriptase.

Still further, the d NTPs is a mixture of d ATP, d TTP, d GTP, and d CTP; the Bst DNA polymerase is a DNA polymerase having both 5 '→ 3' polymerase activity and strand displacement activity.

Still further, in the step 2), the freeze-drying time is 2 hours.

The invention also provides a using method of the sterile carrier obtained by the LAMP reaction system/split system segmentation treatment and normal-temperature transportation method, which comprises the following steps:

a. adding water into a reaction tube containing a sterile carrier of the LAMP reaction system, and dissolving to obtain the LAMP reaction system for LAMP amplification reaction;

or b, adding water into a reaction tube of the sterile carrier containing a plurality of LAMP reaction subsystems, and dissolving to obtain the LAMP reaction system for LAMP amplification reaction.

The invention has the beneficial effects that:

according to the invention, the enzyme component and the PCR reaction solution component are respectively fixed on different sterile carriers (non-woven fabrics or non-woven paper), cut in equal amount and then placed into the reaction tube as required, and the reaction tube is recombined into the one-tube LAMP reaction system, so that the problem of high false positive of the conventional one-tube mixed freeze-drying method is avoided, and the normal-temperature storage or transportation period of the LAMP reaction system is prolonged.

Drawings

FIG. 1 is a display effect diagram of LAMP amplification reaction after sterile vector redissolution of influenza B virus LAMP reaction system;

in the figure, from left to right: the first tube of solution shows pink, and the second tube of solution and the third tube of solution show yellow;

FIG. 2 is a diagram showing the effect of LAMP amplification reaction after the reconstitution of the sterile vector combination of the LAMP reaction subsystem of influenza B virus;

in the figure, from left to right: the first tube of solution showed a dark blue color and the second and third tubes of solution showed a light blue color.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

Example 1

The LAMP reaction system/split system separation treatment and normal temperature transportation method can prepare various LAMP reaction reagent components (including primers, dNTP, Bst polymerase, reaction buffer solution and the like) except for a sample nucleic acid (RNA) to be detected into a one-tube type premixed solution, and prepares an LAMP reaction system with N persons, wherein N is 100, and the preparation method comprises the following steps:

1) preparation of mother liquor

Dd H for primer dry powder₂Dissolving O to prepare a primer working solution for later use;

dd H for trehalose₂Dissolving O to prepare trehalose mother liquor with the trehalose content of 70g per milliliter of water for later use;

2) preparation of LAMP reaction System

The reagent for preparing the LAMP reaction system is 'WarmStart LAMP 2 x premixed solution' of New ENGLAND BioLabs, and comprises d NTPs, Bst polymerase, reaction buffer, dye and other reagent components.

Preparing 100 parts of LAMP reaction system according to the use amount of each component (including primer and WarmStart LAMP 2 multiplied by premixed solution) of each LAMP reaction reagent except for the nucleic acid (RNA) of the sample to be detected in the single-part LAMP reaction system, wherein the total volume is 1.75 mL:

LAMP reaction reagent components	Single dose (mu L)
		WarmStart LAMP 2 Xpremix	12.5
FIP(40pmol/μL)	1
		BIP(40pmol/μL)	1
F3(10pmol/μL)	0.5
		B3(10pmol/μL)	0.5
LF(20pmol/μL)	1
		LB(20pmol/μL)	1
RNA template	1
		H2O	6.5
General System	25

3) Addition of trehalose

Adding the trehalose mother liquor obtained in the step 1) into the LAMP reaction system (with the part N being 100) obtained in the step 2), adding 535.71 mu L of the trehalose mother liquor, adding sterile water to the volume of 2.5mL, and enabling the content of trehalose to be 15g in each milliliter of the volume-fixed mixed liquor.

4) Soaking and freeze-drying of sterile carrier (non-woven fabric or non-woollen paper)

Preparing a sterile carrier according to actual conditions, soaking the sterile carrier in the LAMP reaction system added with the trehalose, uniformly mixing, and carrying out freeze-drying treatment on the sterile carrier for 2 hours by using a freeze dryer.

5) Equivalent cleavage of sterile vector

The lyophilized sterile vector was removed and cut into 100 portions with a cutting instrument in equal amounts.

6) Partial packaging of the System

And subpackaging the cut sterile carrier into reaction tubes, putting one sterile carrier into each tube, and storing or transporting at normal temperature.

The sterile vector containing the LAMP reaction system divided by the method is used for PCR reaction:

1. sample preparation: 1 copy/. mu.L of influenza B virus sample, 10 copies/. mu.L of influenza B virus sample

2. Taking out 3 reaction tubes in the step 6), adding 24 mu L of sterile water into each tube, oscillating and mixing uniformly, adding 1 mu L of sterile water into the first tube as a template, adding 1 mu L of influenza B virus sample with the concentration of 1 copy/mu L into the second tube as a template, adding 1 mu L of influenza B virus sample with the concentration of 10 copies/mu L into the third tube as a template, oscillating and mixing uniformly, and reacting for 30min at 65 ℃ on a PCR instrument.

The reaction results are shown in FIG. 1: the first tube reaction result shows pink and is influenza B virus negative, and the second tube reaction result and the third tube reaction result shows yellow and is influenza B virus positive. The reaction result is accurate, which shows that the LAMP reaction system added with trehalose still has good amplification effect after being frozen and dried and added with water to restore the volume for amplification reaction.

Example 2

In order to avoid the possibility that the one-tube type premix of the LAMP reaction system is possibly influenced by primer dimer to cause false positive after being placed for a long time, and prolong the normal-temperature storage or transportation period of the LAMP reaction system. Various LAMP reaction reagent components except for a sample nucleic acid (DNA) to be detected are prepared according to an LAMP reaction split system, the LAMP reaction split system is an enzyme component system or a PCR reaction solution component system, the enzyme component and the PCR reaction solution component are respectively fixed on different sterile carriers (non-woven fabrics or non-woven paper), and after equal cutting, the enzyme component and the PCR reaction solution component are placed into a reaction tube according to requirements and are recombined into a tube-type LAMP reaction system. At present, LAMP reaction subsystems of N persons are prepared, and the preparation method is as follows, wherein N is 100:

1) preparation of mother liquor

Dd H for primer dry powder₂Dissolving O to prepare a primer working solution for later use;

dd H for trehalose₂Dissolving O to prepare trehalose mother liquor with the trehalose content of 70g per milliliter of water for later use;

2) preparation of LAMP reaction system

a. Preparation of enzyme component system: according to the dosage of Bst DNA polymerase, AMV enzyme and dNTPs in the single-person LAMP reaction system, preparing a 100-person enzyme component system, wherein the total volume is 0.35 mL:

LAMP reaction reagent components	Single dose (mu L)	The dosage of 100 parts (mu L)
			Bst polymerase (8U/. mu.L)	0.75	75
AMV enzyme (8U/. mu.L)	0.25	25
			10×Bst Buffer	2.5	250

b, preparing a PCR reaction solution system: preparing 100 parts of PCR reaction solution system according to the use amounts of the primers, the reaction buffer solution for nucleic acid amplification and other components in the single-part LAMP reaction system, wherein the total volume is 1.65 mL:

LAMP reaction reagent components	Single dose (mu L)	The dosage of 100 parts (mu L)
			dNTPs Mix(2.5mM each)	10	1000
MgSO4(100mM)	0.5	50
			Hydroxynaphthol blue (0.3. mu. mol/mL)	1	100
FIP(40pmol/μL)	1	100
			BIP(40pmol/μL)	1	100
F3(10pmol/μL)	0.5	50
			B3(10pmol/μL)	0.5	50
LF(20pmol/μL)	1	100
			LB(20pmol/μL)	1	100

3) Addition of trehalose

Respectively adding the trehalose mother liquor obtained in the step 1) into the enzyme component system or the PCR reaction solution component system (the part N is 100) obtained in the step 2), adding 160.71 mu L of the trehalose mother liquor into the enzyme component system, and adding sterile water to fix the volume to 0.75 mL; 375 mu L of trehalose mother solution is added into the PCR reaction solution component system, and the volume is adjusted to 2.05mL by adding sterile water.

4) Soaking and freeze-drying of sterile carrier (non-woven fabric or non-woollen paper)

Preparing two sterile carriers according to actual conditions, respectively infiltrating the two sterile carriers into the enzyme component system and the PCR reaction solution system which are added with the trehalose, uniformly mixing, and respectively carrying out freeze drying treatment on the sterile carriers for 2 hours by using a freeze dryer.

5) Equivalent cleavage of sterile vector

Taking out the freeze-dried sterile carrier, and cutting the system infiltrated with the enzyme component or the PCR reaction liquid system into 100 parts by using a cutting instrument in an equivalent manner.

6) Combination and division of systems

And (3) subpackaging the cut sterile carriers (an enzyme component system and a PCR reaction solution component system) into reaction tubes, putting one part of the component system into each tube to form a complete reaction system, and storing or transporting at normal temperature.

The sterile vector containing the LAMP reaction system divided by the method is used for PCR reaction:

1. sample preparation: 1 copy/. mu.L of influenza B virus sample, 10 copies/. mu.L of influenza B virus sample

2. And (3) adding 4 mu L of sterile water into each reaction tube in the step 6), oscillating and mixing uniformly, adding 1 mu L of sterile water into the first tube as a template, adding 1 mu L of influenza B virus sample with the concentration of 1 copy/mu L into the second tube as a template, adding 1 mu L of influenza B virus sample with the concentration of 10 copies/mu L into the third tube as a template, oscillating and mixing uniformly, and reacting for 30min at 65 ℃ on a PCR instrument.

The reaction results are shown in FIG. 2: the first tube shows dark blue and is influenza B virus negative, and the second and third tube shows light blue and is influenza B virus positive. The reaction result is accurate, which shows that the LAMP reaction system added with trehalose still has good amplification effect after being frozen and dried and added with water to restore the volume for amplification reaction.

The products of examples 1-2, the conventional LAMP reaction reagent (liquid) and LAMP reaction reagent (lyophilized powder) were compared as follows:

1. storage and transport mode comparison

Product(s)	Preservation method	Effective period (70% enzyme activity)	Transportation of
				LAMP reaction reagent (liquid state)	-20℃	The loss is small	Freezing
Example 1	At room temperature	80 days	At room temperature
				Example 2	At room temperature	80 days	At room temperature
LAMP reaction reagent (lyophilized powder)	At room temperature	60 days	At room temperature

2. Reaction System establishment time (min)

Product(s)	Reagent thawing	Formulation system
			LAMP reaction reagent (liquid state)	60	30
Example 1	0	1
			Example 2	0	1
LAMP reaction reagent (lyophilized powder)	0	1

3. Specificity of reaction System

In conclusion, compared with the existing LAMP reaction reagent (liquid state), the lyophilized reagent can be transported or stored at normal temperature, and the normal-temperature storage or transportation period of the invention is longer than that of other lyophilization methods; compared with the freeze-dried powder reagent prepared by the conventional LAMP reaction reagent (liquid state) and other freeze-drying methods, the method has the advantages that the enzyme component and the PCR reaction liquid component are respectively fixed on different sterile carriers, are cut in equal amount and then are placed into the reaction tube as required, and are recombined into a tubular LAMP reaction system, so that the problem of high false positive rate of the conventional tubular mixed freeze-drying method is solved, and the detection accuracy is higher.

Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.