CN109735655A

CN109735655A - It is a kind of to detect DNA simultaneously and the multiple fluorescence PCR of RNA prestores freeze-dried reagent

Info

Publication number: CN109735655A
Application number: CN201910055286.6A
Authority: CN
Inventors: 刘白朵; 方琴; 张健; 蒋淼
Original assignee: SHANGHAI KEHUA BIOENGINEERING CO Ltd
Current assignee: SHANGHAI KEHUA BIOENGINEERING CO Ltd
Priority date: 2019-01-21
Filing date: 2019-01-21
Publication date: 2019-05-10

Abstract

The present invention provides a kind of multiple fluorescence PCRs that can detect DNA and RNA simultaneously to prestore freeze-dried reagent, includes reverse transcriptase, archaeal dna polymerase；PCR buffer；Mg²⁺；dNTP；Freeze drying protectant.The present invention can carry out multiple fluorescence PCR augmentation detection for the two distinct types of nucleic acid of DNA and RNA simultaneously in same reaction tube, can solve for different types of nucleic acid, need the case where repeatedly expanding, save time and consumables cost.

Description

It is a kind of to detect DNA simultaneously and the multiple fluorescence PCR of RNA prestores freeze-dried reagent

Technical field

The invention belongs to bioengineering fields, are related to a kind of detection reagent, specifically it is a kind of can detect simultaneously DNA and The multiple fluorescence PCR of RNA prestores freeze-dried reagent.

Background technique

Polymerase chain reaction (Polymerase Chain Reaction), abbreviation PCR is a kind of Amplification Technologies, It, will be to be amplified dependent on the enzymatic reaction of polymerase under the conditions of being existing for template nucleic acid, primer and the deoxynucleotide DNA fragmentation obtains a large amount of specific gene segment through iterative cycles in a short time.Have many advantages, such as sensitive high, high specificity, It is now widely used for diagnostic nucleic acid field.

Fluorescent PCR (Real-time PCR), also known as real-time fluorescence PCR, refer to and fluorescent base are added in PCR reaction system The entire PCR process of real-time monitoring is accumulated using fluorescence signal by group, carries out macroanalysis to unknown template finally by standard curve Or relative quantification is carried out to template by Ct value.Fluorescent PCR can be divided into sonde method and dye method according to the difference of the principles of chemistry.Its In, for Taqman fluorescent quantitation technology based on Taqman fluorescence probe, this probe is an oligonucleotides, at 5 ' ends of sequence It is marked with a fluorescent emission group, a quenching group is marked at 3 ' ends, when probe is complete, emits the fluorescence signal of group Group absorptions are quenched, when PCR amplification, since the circumscribed enzyme effect of Taq enzyme makes probe degrade, to make fluorophor Fluorescence is issued, fluorescent PCR instrument detects sample by detecting the accumulation of fluorescence signal.

Multiple fluorescence PCR (multiplex PCR), also known as multi-primers fluorescent PCR or composite PCR, it is in same PCR Primer, probe in reaction system plus two pairs or more, so that the different nucleic acid fragments to two or more carry out PCR expansion Increase.Certain pathogenic microorganisms, certain hereditary diseases are detected or identified while multiplex PCR is mainly used for a variety of specialized hospital microorganisms.Mesh Before, the multiple fluorescence PCR reagent based on fluorescence probe, which is substantially, detects same type of nucleic acid, is such as all directed to The nucleic acid of DNA class carries out multiple fluorescence PCR, or all carries out multiple fluorescence PCR for the nucleic acid of RNA class.

The fluorescence PCR detection reagent for being conventionally used to DNA and RNA is mostly the substance detection reagent of liquid, a reaction system A target can only be detected, detection project is single, consumptive material and reagent cost are high, and detection flux is low.Simultaneously as being liquid condition Reagent needs cryo-conservation to transport during transportation, increases the cost and complexity of transport.

Current PCR amplification reagent is in liquid condition substantially, is saved at normal temperature and unstable, especially RNA reaction examination Agent.And liquid reagent has a significant impact to the activity of reagent every time using multigelation is required, to the long-distance fortune of PCR reagent It is defeated to will receive very big influence, it is easy that reagent performance is caused to decline because temperature is improper.Of the invention can detect DNA and RNA simultaneously Multiplex PCR pre-freeze reagent can effectively overcome disadvantages mentioned above.

Summary of the invention

For above-mentioned technical problem in the prior art, the multiple of DNA and RNA can be detected simultaneously the present invention provides a kind of Fluorescent PCR prestores freeze-dried reagent, and this multiple fluorescence PCR that can detect DNA and RNA simultaneously prestores freeze-dried reagent and wants Solve the fluorescence PCR detection reagent in the prior art for DNA and RNA can only detect a target/detection project it is single, consumption Material and the technical problem that reagent cost height/detection flux is low and transport is inconvenient.

The present invention provides a kind of multiple fluorescence PCRs that can detect DNA and RNA simultaneously to prestore freeze-dried reagent, comprising following Component:

Reverse transcriptase, the final concentration of 0.02U/ul-0.2U/ul of the reverse transcriptase；

Archaeal dna polymerase, the final concentration of 0.05U/ul-0.1U/ul of the archaeal dna polymerase；

PCR buffer, the PCR buffer is by the KCl of final concentration of 10mM-50mM, final concentration of 5mM-30mM Tris-HCl, whole mass percent concentration are 0.005%-0.05%Tween20 composition；

Mg²⁺, the Mg²⁺Final concentration of 1.5mM-5mM；

The final concentration of 0.1mM-0.5mM of dNTP, the dNTP；

Freeze drying protectant, the freeze drying protectant are dense by the trehalose that whole mass percent concentration is 0.5%-5%, end Spend the D-sorbite for being 5mM-100mM, BSA (bovine serum albumin(BSA)) group that whole mass percent concentration is 0.2%-1.5% At.

The present invention can carry out multi-fluorescence for the two distinct types of nucleic acid of DNA and RNA simultaneously in same reaction tube PCR amplification detection can solve for different types of nucleic acid, need the case where repeatedly expanding, save time and consumptive material Cost.

The present invention is compared with prior art, and technological progress is significant.It is of the invention can freeze-dried reagent can be to two kinds not The pathogen nucleic acid of same type carries out augmentation detection, i.e., detected simultaneously using a reaction system DNA (hepatitis type B virus) and Reagent and detection consumptive material is greatly saved in RNA (Hepatitis C Virus) different types of target.Meanwhile after reagent is lyophilized, Has many advantages, such as convenient transportation, stability is good.

Specific embodiment

Embodiment 1

1) the present invention provides a kind of multiple fluorescence PCRs that can detect DNA and RNA simultaneously to prestore freeze-dried reagent, ingredient Reverse transcriptase containing 0.02U/ul-0.2U/ul, the archaeal dna polymerase of 0.05U/ul-0.1U/ul, the Mg of 1.5mM-5mM²⁺, The dNTP of 0.1mM-0.5mM, 10mM-50mM KCl, 5mM-30mM Tris-HCl, 0.005%-0.05% Tween20, 0.5%-5% trehalose, 5mM-100mM D-sorbite, 0.2%-1.5%BSA.DNA and RNA nucleic acid can be examined simultaneously It surveys.

2) concrete scheme is as follows:

1, experimental group (freeze-dried reagent) formula is as follows:

	Concentration
		KCl	25mM
Tris-HCl	20mM
		Tween20	0.01%
Mg²⁺	3mM
		dNTP	0.2mM
Archaeal dna polymerase	0.05U/ul
		Reverse transcriptase	0.05U/ul
Trehalose	0.5%
		D-sorbite	10mM
BSA	0.5%
		HBV-F	0.2uM
HBV-R	0.2uM
		HBV-P	0.05uM
HCV-F	0.2uM
		HCV-R	0.2uM
HCV-P	0.05uM

After being made into liquid reagent by upper table formula, liquid reagent is put into pre-freeze device, liquid reagent is pre- at -50 DEG C Freeze, is gradually warmed up and carries out vacuum freeze drying.

2, control group (liquid reagent) formula is as follows:

Oscillation mixes, and is dispensed into 8 company, 96 orifice plate.

HBV-F sequence are as follows: 5 '-tcgcatcaggactcctaggacc-3 ' (shown in SEQ ID NO.1)；

HBV-R sequence are as follows: 5 '-cgagtctagactctgtggtattgtgagg-3 ' (shown in SEQ ID NO.2)；

HBV-P probe sequence are as follows: 5 '-CY5-ctgctcgtgttacaggcggggtttttcttg-BHQ3-3 ' (SEQ ID Shown in NO.3)；

HCV-F sequence are as follows: 5 '-ctagccatggcgttagtatgagtgt-3 ' (shown in SEQ ID NO.4)；

HCV-R sequence are as follows: 5 '-tggcaattccggtgtactcac-3 ' (shown in SEQ ID NO.5)；

HCV-P probe sequence are as follows: 5 '-FAM-cgggagagccatagtggtctgcggaa-BHQ1-3 ' (SEQ ID NO.6 It is shown)；

3, DNA and RNA is extracted from clinical sample blood plasma, is added separately to the good experimental group of above-mentioned packing and control group examination In agent.

4, setting program is as follows:

50 DEG C, 25min；95 DEG C, 10min；

5 × (95 DEG C, 15S；55 DEG C, 20S；72 DEG C, 20S；)

45 × (95 DEG C, 10S；60 DEG C, 45S；Acquire fluorescence；)

5, experimental group and control group testing result are as follows:

5.1 Table A DNA testing results

Group number	Sensitivity
		Experimental group	10IU
Control group	10IU

Table B RNA testing result

Group number	Sensitivity
		Experimental group	50IU
Control group	50IU

By Table A, table B it is found that experimental group (freeze-dried reagent) and contrast agents (liquid reagent) detection sensitivity are almost the same, Freeze-dried reagent is able to maintain the high sensitivity of liquid reagent.

The advantage of the invention is that detection reagent can detect the two distinct types of nucleic acid of DNA and RNA simultaneously, it is able to achieve pair The detection of multiple nucleic acids is greatly saved kit detection consumptive material, expands application range.The freeze-dried reagent that prestores of the invention exists After frozen dried, reagent is allowed to have many advantages, such as convenient transportation, stability is good.

Claims

DNA can be detected simultaneously and the multiple fluorescence PCR of RNA prestores freeze-dried reagent 1. a kind of, it is characterised in that include following component:

Reverse transcriptase, the final concentration of 0.02U/ul-0.2U/ul of the reverse transcriptase；

Archaeal dna polymerase, the final concentration of 0.05U/ul-0.1U/ul of the archaeal dna polymerase；

PCR buffer, the PCR buffer is by the KCl of final concentration of 10mM-50mM, final concentration of 5mM-30mM Tris-HCl, whole mass percent concentration are 0.005%-0.05% Tween20 composition；

Mg²⁺, the Mg²⁺Final concentration of 1.5mM-5mM；

The final concentration of 0.1mM-0.5mM of dNTP, the dNTP；

Freeze drying protectant, the trehalose, final concentration of that the freeze drying protectant is 0.5%-5% by whole mass percent concentration The BSA that the D-sorbite of 5mM-100mM, whole mass percent concentration are 0.2%-1.5% is formed.