CN102994649A - Multi-gene detection method of rash pathogens based on capillary electrophoresis - Google Patents

Abstract

The invention discloses a multi-gene detection method of rash pathogens based on capillary electrophoresis. The multi-gene detection method comprises the following steps: (1) producing a kit for multi-gene detection of the rash pathogens; (2) collecting a sample from a patient and extracting nucleic acid; (3) taking the nucleic acid of the patient as a template for performing reverse transcription; (4) taking a reverse transcription product as the template for performing PCR (polymerase chain reaction) reaction; and (5) separating the sample through a capillary electrophoresis method. The multi-gene detection method disclosed by the invention has the advantages of high sensitivity, good repeatability, strong accuracy, strong flexibility and low cost.

Description

A kind of multiple gene tester of the eruption venereal disease substance based on electrocapillary phoresis

Technical field

The present invention relates to a kind of detection method, especially a kind of highly sensitive, good reproducibility, accuracy is strong, handiness is strong, cost is low based on the multiple gene tester of the eruption venereal disease substance of electrocapillary phoresis.

Background technology

At present, China's eruption venereal disease substance detects main method has following several:

1. pathogenic examination: namely directly do image and examination of living tissue, or pathogen isolation cultivates, under microscope, Electronic Speculum, observe afterwards, make diagnosis.This method once once had been the classical way of microorganism detection, also be after the basic platform of various detection methods, obtain the approval of every country, use till today in China always.Advantage: cost is low; Low to the laboratory hardware requirement.Shortcoming: (1) length consuming time: generally need 3-5 days or longer.(2) diagnosis efficiency is low: can only single bacterium pure culture; Pathogenic agent to some phenotypic characteristic variations is difficult to distinguish with the morphology experience; In addition, because antibiotic abuse, bacterium grows in testing process and is suppressed, and causes false-negative result.(3) workload is huge: owing to must detect every kind of bacterium of each sampling observation sample, workload is very huge, and particularly part requires comparatively harsh bacterium to culture environment, has more strengthened the workload and the difficulty that detect.

2. immunologic test: most widely used is Enzyme-multiplied immune technique (ELISA): generally use first primary antibodie and antigen generation specific binding, then with two anti-and primary antibodie generation specific bindings of general enzyme labelling, enzyme is developed the color, afterwards observations again.Advantage: (1) sample flux is higher: utilize 96 hole enzyme plates, one flat plate can be finished the detection of a plurality of samples; (2) responsive: as to utilize the characteristic of enzyme connection, original antigen signals can be amplified; (3) quick: as in time, will to shorten much than traditional substratum microorganism culturing test procedure.Shortcoming: (1) is prone to false positive: owing to wash and antigen coated operation, or cross reaction occurs owing to the antigenic surface determinant is similar, so be prone to false positive (2); Take time and effort: making antibody is the work (3) that takes time and effort very much; Sensitivity is uncertain: the quality of antibody is depended in the sensitivity of experiment, and the quality of each antibody differs, difficult quality control; (4) can't detect the virus of multi-Vari: variability is virus faster, such as first stream virus, has multiple hypotype, and often variation, and variation causes antibody to lose efficacy, can't detectable antigens, and such as avian influenza virus and swine influenza virus etc.

3. molecular biology-PCR detection method.That uses at present often has real-time fluorescence quantitative PCR, immuno-PCR, a reverse transcription PCR etc., all is that the specific target gene for pathogenic agent detects.Wherein, fluorescent quantitative PCR detection method is the most ripe.The advantage of fluorescent quantitative PCR technique: susceptibility is high, but and accurate quantification, in the detection to Listeria monocytogenes, Salmonellas, Clostridium botulinum, intestinal bacteria etc., all obtained good result.2004, China issued the national standard of implementing fluorescence quantitative PCR detection bird flu H7 hypotype.2009, drugs approved by FDA with the test kit of fluorescence quantitative PCR detection porcine influenza H1N1 hypotype.The shortcoming of quantitative fluorescent PCR: (1) flux is low: once can only detect a cause of disease composition, detect multiple pathogenic bacteria such as needs, just need multiple pc R instrument to work simultaneously, efficient is low, cycle is long, and the sample of large batch of multiple pathogen contamination is had no way of doing it especially; (2) cost is relatively high: because once detecting a pathogenic agent, when a sample needs to detect multiple pathogenic bacteria simultaneously, must detect one by one, cost increases relatively.

4. molecular biology-gene chips: gene chip is to pass through micro-processing technology, dna fragmentation (gene probe) with the particular sequence of ten hundreds of and even 1,000,000, arrange regularly and be fixed on the upholders such as silicon chip, slide, a two-dimentional dna probe array that consists of, utilize the biological sample of this class chip and mark to hybridize, can carry out fast qualitative and quantitative analysis to the gene expression profile bioinformation of sample.Advantage: (1) high-flux parallel detects: when a sample needed to detect multiple pathogenic bacteria simultaneously, once experiment can draw whole results; (2) easy and simple to handle quick: whole detection only needed 4-8 hour substantially can go out the result.Shortcoming: (1) technical costs is expensive, complicated: each sample needs a chip, and cost Da Yu $1000/ sample is unfavorable for large-scale promotion; Synthetic and the fixing more complicated of probe is particularly made highdensity probe array, is main rate-limiting step; (2) can not accurate quantification, poor repeatability; (3) sensitivity is lower: chip method needs the nucleic acid amount larger, generally must do first the multiplex PCR amplification, because primer is more, self produces dimer easily, hairpin structure, or because the Tm value is different, and the purpose fragment efficient that causes increasing is different, and then the sensitivity of impact detection; (4) because the kind of chip is more, be difficult to formulate a unified quality control standard.

Summary of the invention

The purpose of this invention is to provide a kind of highly sensitive, good reproducibility, accuracy is strong, handiness is strong, cost is low based on the multiple gene tester of the eruption venereal disease substance of electrocapillary phoresis.

The present invention adopts following technical scheme:

A kind of multiple gene tester of the eruption venereal disease substance based on electrocapillary phoresis comprises the steps: that (1) produces " the multiple gene detecting kit of eruption venereal disease substance "; (2) gather patient's sample, and extract nucleic acid; (3) carry out reverse transcription take patient's nucleic acid as template; (4) carry out the PCR reaction take reverse transcription product as template; (5) with the method sample separation of electrocapillary phoresis.

Preferably, the test kit in the described step (1) comprises: reverse transcription primer pipe, PCR primer pipe, reverse transcription damping fluid, PCR damping fluid, 25mM magnesium chloride, ThermoScript II, archaeal dna polymerase, fluorescent marker, positive control.

Preferably, the patient's sample in the described step (2) comprises Nasopharyngeal swabs, sputum, bleb liquid, blood etc.

Preferably, described step (3) comprises the substep of hatching at a certain temperature in proportion behind sample panel adding reagent and sample and mixing.

Preferably, the incubation temperature in the described step (3) is respectively 48 ℃, 42 ℃, 95 ℃, 4 ℃.

Preferably, described step (4) comprises the substep that carries out in proportion thermal cycle reaction after sample panel adds reagent and sample and mixing by certain temperature.

Preferably, the thermal cycling temperature in the substep of described step (4) is respectively 95 ℃, 94 ℃, 60 ℃, 70 ℃, 4 ℃.

Beneficial effect of the present invention is:

1. highly sensitive, good reproducibility: adopt laser induced fluorescence(LIF)-PMT, have hypersensitivity.

2. accuracy is strong: adopt capillary electrophoresis that the PCR product is carried out separation detection, non-specific amplification product, primer dimer and specific amplification products can be separated, at utmost reduce false positive.

3. high-throughput: native system adopts two (96 hole) plates, automatic sample and sample tracer technique, realize a single reaction detection 15-40 site, can do simultaneously 192 reactions (such as 192 patient's samples, 14 kinds of venereal disease substances of paying a home visit of each sample detection, 23 sites), went out the result in one day; For co-infected patients, present method can disposablely provide accurate report, avoids undetected.

4. accurate quantification: but accurate quantification pathogen gene copy number.

5. handiness is strong: the target gene that can adjust according to demand at any time detection.

6. cost is low: the testing cost Shao Yu $50 of each sample is beneficial to large-scale promotion.

Embodiment

The below is described in further detail the present invention according to embodiment.

The present invention has founded the multiple gene test scheme of 16 kinds of common eruption venereal disease substances of a kind of synchronous detection, 22 target position.

The virus that detects comprises herpes simplex virus 1, herpes simplex virus type 2, varicella zoster virus, epstein-barr virus (EB), the human cytomegalovirus, human herpes 6 C-type virus Cs, human herpes 7 C-type virus Cs, enterovirus and hypotype thereof (coxsackie virus A 16-type and enterovirns type 71), Measles virus, rubella virus, assays for parvovirus B 19, foot and mouth disease virus and hypotype thereof (foot and mouth disease virus A type, O type, C type, Asia type, SAT1,2,3), beta hemolytic streptococcus, mycoplasma pneumoniae etc. (table 1).

Set up the contrast (table 1) of reliable sample quality and reaction process

A) the contrast confidential reference items of people DNA and people RNA integrity: guarantee in checkout procedure false negative is avoided in the judgement of sample quality.

B) normal reaction contrast confidential reference items: monitoring PCR reaction efficiency, avoid false negative.The design of primers of the multiple gene test of eruption venereal disease substance (seeing Table 2 nucleotide sequences)

The target site that table 1. detects

Table 2: nucleotides sequence tabulation

Concrete steps are as follows:

1. produce " the multiple gene detecting kit of eruption venereal disease substance ", comprise in the test kit:

1) reverse transcription damping fluid;

2) reverse transcription primer pipe (RT primer mix);

3) ThermoScript II;

4) PCR damping fluid;

5) PCR primer pipe (PCR primer mix);

6) fluorescent marker;

7) 25mM magnesium chloride (MgCl ₂);

8) archaeal dna polymerase (Taq DNAPolymerase);

9) positive control (Positive Control).

2. gather patient's sample (Nasopharyngeal swabs, sputum, bleb liquid, blood etc.), and extract nucleic acid

3. carry out reverse transcription (RT) take patient's nucleic acid as template:

1) add reagent and sample (RT plate) in following ratio in 96 hole sample panel:

Annotate: positive control: 1 μ L/ reaction

2) hatch by following temperature behind the mixing:

Step	Temperature	Time
			1	48℃	1 minute
2	42℃	60 minutes
			3	95℃	5 minutes
4	4℃	Continue: until collect the RT product

4. carry out the PCR reaction take reverse transcription product as template

1) add reagent and sample (PCR plate) in following ratio in 96 hole sample panel:

The PCR reaction reagent	Amount/hole
		The PCR damping fluid, 5X	4μL
25mM MgCl2	4μL
		The PCR primer	2μL
Archaeal dna polymerase	0.7μL
		The RT product	9.3μL
Total	20μL

2) carry out thermal cycle reaction by following temperature behind the mixing:

Step	Temperature	Time
			1	95℃	10 minutes
2	94℃	30 seconds
			3	60℃	30 seconds
4	70℃	1 minute
			5	N/A	Repeat 2-4 step 34 time (totally 35 times)
6	70℃	1 minute
			7	4℃	Continue: until collect the PCR product

5.GenomeLab GeXP genetic analyzer electrocapillary phoresis sample separation

1) preparation GeXP sample:

The GeXP sample	Amount/hole
		The SLS sample solution	38.75μL
DNA size criteria 400	0.5μL
		The PCR product	1μL
Total	40μL
		Mineral oil	1

Annotate: PCR product amount can be 0.1-1 μ L, or water dilutes rear loading on demand in advance

2) prepare parting liquid: about 220 μ L parting liquids are added in the hole of proper number on the 96 hole parting liquid plates;

3) electrocapillary phoresis sample separation.

6. interpretation of result.

Obviously, the above embodiment of the present invention only is for example of the present invention clearly is described, and is not to be restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make on the basis of the above description other multi-form variation and changes.Here can't give all embodiments exhaustive.Everyly belong to the row that apparent variation that technical scheme of the present invention amplifies out or change still are in protection scope of the present invention.

Claims (7)

1. the multiple gene tester based on the eruption venereal disease substance of electrocapillary phoresis comprises the steps:

(1) produces " the multiple gene detecting kit of eruption venereal disease substance ";

(2) gather patient's sample, and extract nucleic acid;

(3) carry out reverse transcription take patient's nucleic acid as template;

(4) carry out the PCR reaction take reverse transcription product as template;

(5) with the method sample separation of electrocapillary phoresis.

2. detection method according to claim 1, the test kit in the described step (1) comprises: reverse transcription primer pipe, PCR primer pipe, reverse transcription damping fluid, PCR damping fluid, 25mM magnesium chloride, ThermoScript II, archaeal dna polymerase, fluorescent marker, positive control.

3. detection method according to claim 1, the patient's sample in the described step (2) comprises Nasopharyngeal swabs, sputum, bleb liquid, blood etc.

4. detection method according to claim 1, described step (3) comprise in proportion and add the substep of hatching at a certain temperature behind reagent and sample and the mixing in sample panel.

5. detection method according to claim 4, the incubation temperature in the described step (3) is respectively 48 ℃, 42 ℃, 95 ℃, 4 ℃.

6. detection method according to claim 1, described step (4) comprise the substep that carries out in proportion thermal cycle reaction after sample panel adds reagent and sample and mixing by certain temperature.

7. detection method according to claim 6, the thermal cycling temperature in the substep of described step (4) is respectively 95 ℃, 94 ℃, 60 ℃, 70 ℃, 4 ℃.