CN101974634B

CN101974634B - Toxoplasma gondii rapid detection kit and preparation method thereof

Info

Publication number: CN101974634B
Application number: CN 201010526397
Authority: CN
Inventors: 张德林; 王萌; 王艳华; 付宝权; 李文卉
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2010-10-30
Filing date: 2010-10-30
Publication date: 2012-08-01
Anticipated expiration: 2030-10-30
Also published as: CN101974634A

Abstract

The invention relates to a Toxoplasma gondii rapid detection kit and a preparation method thereof. The kit is based on the molecular biology and the nucleoside triphosphate hydrolase (NTPase) gene with high conservation of Toxoplasma gondii to adopt the loop-mediated isothermal amplification (LAMP) method to perform the rapid detection of Toxoplasma gondii. The kit contains LAMP reaction solution, primer, Bst DNA polymerase, color reagent, standard positive template and dd H2O. The kit of the invention is characterized by high sensitivity and specificity, rapid reaction, simple operation and the like, does not require special instrument and equipment and solves the defects of the prior art such as long detection time of toxoplasmosis, large workload and complicated operation. The kit of the invention can be used to detect whether a clinical sample contains Toxoplasma gondii and used in the early diagnosis and the molecular epidemiological study of toxoplasmosis.

Description

A kind of toxoplasma gondii quick detection kit and preparation method thereof

Technical field

The present invention relates to detect the loop-mediated isothermal amplification technique of toxoplasma cdna group DNA, is a kind of toxoplasma gondii quick detection kit specifically, the present invention includes the preparation method of this test kit.

Background technology

The method of diagnosis toxoplasmosis commonly used comprises agglutination test, enzyme-linked immunosorbent assay and PCR etc. at present, and these methods all have certain shortcoming.Agglutination test mainly is an indirect hemagglutination method, and the shortcoming of this method is that detection sensitivity is low, the situation of omission can occur; The shortcoming of enzyme-linked immunosorbent assay is that required plant and instrument is expensive, and it is long to be used special ELIASA, operating process relative complex, detection time; And these two kinds of methods are that the animal of test positive possibly inoculate the toxoplasma gondii vaccine and cause to the toxoplasma antibody detection.PCR and LAMP method all are based on the sensitivity on the molecular biology basis, special, new high-efficiency diagnostic techniques.But PCR method needs expensive PCR appearance to be used; The time of reaction is long; Need carry out agarose gel electrophoresis after the reaction with gel imaging appearance observations; And in animal gene group DNA extraction process, the supressor that possibly have some PCR that leaves over reactions influences its specificity and susceptibility.

Summary of the invention

The objective of the invention is in order to overcome the deficiency in the current toxoplasmosis diagnostic techniques; Provide a kind of based on biology field hypersensitivity, high specific, high efficiency, toxoplasma gondii quick detection kit simple to operate, the preparation method of this test kit is provided simultaneously.

A kind of toxoplasma gondii quick detection kit comprises 2 * LAMP reaction solution, primer mixture, big fragment strand displacement archaeal dna polymerase, developer, standard positive template and dd H ₂O.Said primer mixture comprises: the inner primers F IP in the 40pmol upper reaches, the inner primer BIP in 40pmol downstream, the outside primers F 3 in the 5pmol upper reaches, the outside primer B3 in 5pmol downstream, 20pmol upper reaches annular primer LF and 20pmol downstream annular primer LB.

Said primer is to toxoplasma gondii research of nucleoside triphosphate hydrolase (NTPase) gene design, and primer sequence is:

The inner primers F IP (F1c+F2) in the upper reaches:

5′-ACCTAGCTGGGTCTTCGCCTAG-TTTT-GCCTCTTCGCGTTTATCACG-3′

The outside primer BIP (B1c+B2) in downstream:

5′-AATACGGGGTGAAGCATTGCCG-TTTT-AGAAGCACCCCCAACCTC-3′

The outside primers F 3:5 ' in the upper reaches-CGATCACAGGAGCTGAGGA-3 '

The outside primer B3:5 ' in downstream-CGGTACCTTCCTGTAGTGGA-3 '

Upper reaches ring primer LF:5 '-AAAGAGGGCGTCGCGGTAC-3 '

Downstream ring primer LB:5 '-GAGGAAGGCCTCTTCGCGTTTATC-3 '

Said 2 * LAMP reaction solution comprise 4% trishydroxymethyl ethylamine (Tris-HCl, pH8.8), 20mM Repone K (KCl), 16mM sal epsom (MgSO ₄), 20mM ammonium sulfate ((NH4) ₂SO ₄), 0.2% polysorbas20 (Tween20), 2.8mM deoxynucleoside acid mixture (dNTPs) and 1.6M trimethyl-glycine.

The preparation method of said toxoplasma gondii quick detection kit is following:

(1) preparation 2 * LAMP reaction solution: 1. dispose Tris-HCl: configuration concentration is 12.11%Tris, and regulates pH8.8 with HCl, and is subsequent use; 2. to contain final concentration be the KCl of 22.2mM, the MgSO of 17.8mM in configuration ₄, 22.2mM (NH4) ₂SO ₄With the mixing solutions of the trimethyl-glycine of 1.78M, and add the Tris-HCl of 0.22%Tween20 and 4.4%, subsequent use; 3. the solution of configuration and the mixed that 25mM dNTPs according to volume ratio is 9: 1 in inciting somebody to action 2.;

(2) preparation primer mixture: the zone design primer that 1. is directed against one section high conservative of toxoplasma gondii research of nucleoside triphosphate hydrolase gene; 2. FIP in the mix primer: BIP: F3: B3: LF: LB is 8: 8: 1: 1: 4: 4, and according to as above volume ratio mixing.

(3) preparation standard positive template: 1. body weight is that every of 20～25g mouse is by 1 * 10 ⁵～1 * 10 ⁶Toxoplasma tachyzoite abdominal cavity inoculation, through 3～4 days, mouse occur spirit depressed, do not eat, during by the thick symptom such as disorderly of hair, take off the neck small white mouse that causes death, be soaked in body surface sterilization in 70～75% alcohol; 2. every mouse is collected polypide with 2 milliliters of PH7.2PBS damping fluid flushing abdominal cavities; 3. extract toxoplasma cdna group DNA, this is a standard positive template.

Reaction system is 50%2 * LAMP reaction solution, 3.6% primer mixture, 0.4%BstDNA polysaccharase, 8% standard positive template and 34.4%dd H ₂O.Reaction conditions is 65 ℃, 30min; 80 ℃, 2min; 4 ℃, the permanent preservation.Be the LAMP product to be mixed with developer in 25: 1 by volume after the reaction, observe colour-change, judged result.

The present invention is based upon on the molecular biology basis; Research of nucleoside triphosphate hydrolase gene based on the toxoplasma gondii high conservative; Use the test kit of the method rapid detection toxoplasma gondii of ring mediated isothermal amplification (LAMP); Characteristics such as having susceptibility height, high specificity, be swift in response, be simple to operate do not need special plant and instrument, have solved toxoplasmosis long, defectives such as workload is big, complicated operation detection time; Can be used to detect clinical sample and whether carry toxoplasma gondii, and the early diagnosis of toxoplasmosis and Molecule Epidemiology Investigation.

Embodiment

A kind of toxoplasma gondii quick detection kit comprises: 2 * LAMP reaction solution, primer mixture, BstDNA polysaccharase, developer, standard positive template and dd H ₂O.

2 * LAMP reaction solution comprises: 4%Tris-HCl (pH8.8), 20mM KCl, 16mM MgSO ₄, 20mM (NH4) ₂SO ₄, 0.2%Tween 20,2.8mM dNTPs and 1.6M trimethyl-glycine.

Primer mixture comprises:

The inner primers F IP in the 40pmol upper reaches, the inner primer BIP in 40pmol downstream, the outside primers F 3 in the 5pmol upper reaches, the outside primer B3 in 5pmol downstream, 20pmol upper reaches annular primer LF and 20pmol downstream annular primer LB.

Below in conjunction with embodiment the present invention is explained further details:

Embodiment 1

(1) preparation LAMP reaction solution:

1. dispose Tris-HCl: take by weighing 121.1g Tris in 800mLddH ₂Dissolve among the O, regulate pH8.8, be settled to 1000mL with HCl, subsequent use;

2. take by weighing 1.49g KCl, 3.96g MgSO ₄7H ₂O, 2.64g (NH4) ₂SO ₄, the 187.4g trimethyl-glycine, and add 40mL 1. in the Tris-HCl and the 2mL Tween 20 of configuration, add dd H ₂O is settled to 900mL, and is subsequent use;

3. get the 2. solution of middle configuration of 900 μ L, add 100 μ L25mM dNTPs, mixing.

(2) preparation primer mixture:

1. be directed against the zone of one section high conservative of toxoplasma gondii research of nucleoside triphosphate hydrolase gene, utilize the online software design primer of PrimerExpolorer V4;

2. mix primer comprises 22.4 μ L FIP and BIP, 2.8 μ L F3 and B3 and 11.2 μ L LF and LB, mixing.

(3) preparation standard positive template:

1. 20～25 every of gram body weight mouse press 1 * 10 ⁵～1 * 10 ⁶Toxoplasma tachyzoite abdominal cavity inoculation, through 3～4 days, mouse occur spirit depressed, do not eat, during by the thick symptom such as disorderly of hair, take off the neck small white mouse that causes death, be soaked in body surface sterilization in 70～75% alcohol;

2. every mouse is collected polypide with 2 milliliters of PH7.2PBS damping fluid flushing abdominal cavities;

3. extract toxoplasma cdna group DNA, this is a standard positive template.

During detection according to following reaction system

LAMP reaction solution 12.5 μ L

BstDNA polysaccharase 1 μ L

Primer mixture 0.9 μ L

Template DNA 2 μ L

dd?H ₂O 8.6μL

Total reaction system 25 μ L

(4) LAMP reaction

Reaction conditions is 65 ℃, 30min; → 80 ℃, 2min; → 4 ℃, the permanent preservation.

Reaction result is judged: the reaction back adds the developer of 1 μ L, and the visual inspection positive findings is that dyestuff becomes green by orange, and ultraviolet issues green fluorescence; The nondiscoloration of negative findings dyestuff still is an orange, does not have fluorescence under the ultraviolet.

Claims

1. a toxoplasma gondii quick detection kit comprises 2 * LAMP reaction solution, primer mixture, BstDNA polysaccharase, developer, standard positive template and dd H ₂O; It is characterized in that said primer mixture is to toxoplasma gondii research of nucleoside triphosphate hydrolase gene design; Comprise: the inner primers F IP in the 40pmol upper reaches, the inner primer BIP in 40pmol downstream, the outside primers F 3 in the 5pmol upper reaches, the outside primer B3 in 5pmol downstream, 20pmol upper reaches annular primer LF and 20pmol downstream annular primer LB, said primer sequence is:

The inner primers F IP (F1c+F2) in the upper reaches:

5′-ACCTAGCTGGGTCTTCGCCTAG-TTTT-GCCTCTTCGCGTTTATCACG-3′

The outside primer BIP (B1c+B2) in downstream:

5′-AATACGGGGTGAAGCATTGCCG-TTTT-AGAAGCACCCCCAACCTC-3′

The outside primers F 3:5 ' in the upper reaches-CGATCACAGGAGCTGAGGA-3 '

The outside primer B3:5 ' in downstream-CGGTACCTTCCTGTAGTGGA-3 '

Upper reaches ring primer LF:5 '-AAAGAGGGCGTCGCGGTAC-3 '

Downstream ring primer LB:5 '-GAGGAAGGCCTCTTCGCGTTTATC-3 '.

2. toxoplasma gondii quick detection kit according to claim 1 is characterized in that said 2 * LAMP reaction solution comprises the Tris-HCl of 4%pH8.8,20mM KCl, 16mM MgSO ₄, 20mM (NH4) ₂SO ₄, 0.2%Tween 20,2.8mM dNTPs and 1.6M trimethyl-glycine.

3. preparation method according to the said toxoplasma gondii quick detection kit of claim 1 is characterized in that by following process method preparation:

A, preparation 2 * LAMP reaction solution: 1. dispose Tris-HCl: configuration concentration is 12.11%Tris, and regulates pH8.8 with HCl, and is subsequent use; 2. to contain final concentration be the KCl of 22.2mM, the MgSO of 17.8mM in configuration ₄, 22.2mM (NH4) ₂SO ₄With the mixing solutions of the trimethyl-glycine of 1.78M, and add the Tris-HCl of 0.22%Tween20 and 4.4%, subsequent use; 3. the solution of configuration and the mixed that 25mM dNTPs according to volume ratio is 9: 1 in inciting somebody to action 2.;

B, preparation primer mixture: the zone design primer that 1. is directed against one section high conservative of toxoplasma gondii research of nucleoside triphosphate hydrolase gene; 2. FIP in the mix primer: BIP: F3: B3: LF: LB is 8: 8: 1: 1: 4: 4, and according to as above volume ratio mixing.

C, preparation standard positive template: 1. 20～25 every of gram body weight mouse press 1 * 10 ⁵～1 * 10 ⁶Toxoplasma tachyzoite abdominal cavity inoculation, through 3～4 days, mouse occur spirit depressed, do not eat, during by the thick symptom such as disorderly of hair, take off the neck small white mouse that causes death, be soaked in body surface sterilization in 70～75% alcohol; 2. every mouse is collected polypide with 2 milliliters of PH7.2PBS damping fluid flushing abdominal cavities; 3. extract toxoplasma cdna group DNA, this is a standard positive template.