CN102994650B - Multi-gene detection method of encephalitis viruses based on capillary electrophoresis - Google Patents

Abstract

The invention discloses a multi-gene detection method of encephalitis viruses based on capillary electrophoresis. The multi-gene detection method comprises the following steps: (1) producing a kit for multi-gene detection of the encephalitis viruses; (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 ofly cause encephalitis multiple gene detection method based on electrocapillary phoresis

Technical field

The present invention relates to a kind of detection method, especially a kind of highly sensitive, reproducible, accuracy is strong, handiness is strong, cost is low based on electrocapillary phoresis causes encephalitis multiple gene detection method.

Background technology

At present, China causes encephalitis detection main method has several as follows:

1. pathogenic examination: namely directly do image and examination of living tissue, or pathogen isolation is cultivated, and afterwards at microscope, electric Microscopic observation, makes diagnosis.This method had once been once the classical way of microorganism detection, and the basic platform of various detection method after being also, obtains the accreditation of every country, use till today in China always.Advantage: cost is low; Low to 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; To the pathogenic agent that some phenotypic characteristics make a variation, be difficult to distinguish by morphology experience; In addition, due to antibiotic abuse, bacterium grows and is suppressed in testing process, causes false-negative result.(3) workload is huge: owing to must detect often kind of bacterium of each sampling observation sample, workload is very huge, and particularly part requires comparatively harsh bacterium to culture environment, more increases workload and the difficulty of detection.

2. immunologic test: most widely used is Enzyme-multiplied immune technique (ELISA): generally first use primary antibodie and antigen generation specific binding, then with two anti-and primary antibodie generation specific bindings of general enzyme labelling, again enzyme is developed the color, observations afterwards.Advantage: (1) sample throughput is higher: utilize 96 hole enzyme plates, one flat plate can complete the detection of multiple sample; (2) more responsive: to utilize the characteristic that enzyme joins, original antigen signals can be amplified; (3) quick: in time, will to shorten much than traditional substratum microorganism culturing test procedure.Shortcoming: (1) easily occurs false positive: due to washing and antigen coated operation, or due to antigenic surface determinant similar and cross reaction occurs, therefore easily there is false positive; (2) take time and effort: making antibody is the work taken time and effort very much; (3) sensitivity is uncertain: the quality of antibody is depended in the sensitivity of experiment, and the quality of each antibody differs, and difficult quality controls; (4) virus of multi-Vari cannot be detected.

3. molecular biology-PCR detection method.At present often application has real-time fluorescence quantitative PCR, immuno-PCR, reverse transcription PCR etc., is all to detect for the specific target gene of pathogenic agent.Wherein, fluorescent quantitative PCR detection method is the most ripe.The advantage of fluorescent quantitative PCR technique: susceptibility is high, and can accurate quantification, in the detection to Listeria monocytogenes, Salmonellas, Clostridium botulinum, intestinal bacteria etc., all achieve good result.2004, China issued the national standard implementing fluorescence quantitative PCR detection bird flu H7 hypotype.2009, U.S. FDA have approved the test kit by 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, as needs detect multiple pathogenic bacteria, just need multiple stage PCR instrument to work, efficiency is low simultaneously, cycle is long, has no way of doing it especially to the sample of large batch of multiple pathogen contamination; (2) cost is relatively high: because once detecting a pathogenic agent, and 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 passes through micro-processing technology, by ten hundreds of and even 1,000,000 the DNA fragmentation (gene probe) of particular sequence, arrangement is fixed on the upholder such as silicon chip, slide regularly, the two-dimentional DNA probe array formed, utilize the biological sample of this kind of chip and mark to hybridize, fast qualitative and quantitative analysis can be carried out to the gene expression profile bioinformation of sample.Advantage: (1) high-flux parallel detects: when a sample needs to detect multiple pathogenic bacteria simultaneously, once experiment can draw whole result; (2) fast easy and simple to handle: whole detection only needs 4-8 hour substantially can go out result.Shortcoming: (1) technical costs is expensive, complicated: each sample needs a chip, and cost great Yu $1000/ sample, is unfavorable for large-scale promotion; The synthesis of probe and fixing more complicated, particularly make highdensity probe array, is main rate-limiting step; (2) can not accurate quantification, poor repeatability; (3) sensitivity is lower: chip method needs nucleic acid amount comparatively large, generally first must do multiplexed PCR amplification, because primer is more, easily self produces dimer, hairpin structure, or due to Tm value difference, and cause amplification object fragment efficiency different, and then the sensitivity that impact detects; (4) kind due to chip is more, is difficult to the quality control standard that formulation one is unified.

Summary of the invention

The object of this invention is to provide a kind of highly sensitive, reproducible, accuracy is strong, handiness is strong, cost is low multiple gene detection method causing encephalitis based on electrocapillary phoresis.

The present invention adopts following technical scheme:

Based on the multiple gene detection method causing encephalitis of electrocapillary phoresis, comprise the steps: that (1) produces " causing encephalitis multiple gene detection kit "; (2) gather Patient Sample A, and extract nucleic acid; (3) with patient's nucleic acid for template carries out reverse transcription; (4) be that template carries out PCR reaction with reverse transcription product; (5) with the method sample separation of electrocapillary phoresis.

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

Preferably, the Patient Sample A in described step (2) comprises blood, cerebrospinal fluid etc.

Preferably, described step (3) comprise in sample panel, add reagent and sample and mixing in proportion after the sub-step of hatching at a certain temperature.

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

Preferably, described step (4) comprise in sample panel, add reagent and sample and mixing in proportion after to carry out the sub-step of thermal cycle reaction by certain temperature.

Preferably, the thermal cycling temperature in the sub-step of described step (4) is respectively 95 DEG C, 94 DEG C, 60 DEG C, 70 DEG C, 4 DEG C.

Beneficial effect of the present invention is:

1. highly sensitive, reproducible: to adopt laser induced fluorescence(LIF)-PMT, there is hypersensitivity.

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

3. high-throughput: native system adopts two (96 hole) plate, automatic sample and sample tracer technique, realize a single reaction detection 15-40 site, 192 reactions can be done (as 192 Patient Sample A simultaneously, each sample detection 14 kinds of pathogen of paying a home visit, 23 sites), within one day, go out result; For co-infected patients, present method disposablely can provide accurate report, avoids undetected.

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

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

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

Embodiment

According to embodiment, the present invention is described in further detail below.

The virus detected comprises encephalitis b virus, fores encephalitis virus, Bo Wasen virus, west nile virus, hsv (I type and II type), varicella zoster virus, human cytomegalovirus, epstein-barr virus (EB), human herpes 6 C-type virus C, human herpes 7 C-type virus C, all enteroviruses, coxsackie virus A 16-type, enterovirns type 71, mumps virus, Measles virus, Nipah virus, rubella virus, rabies virus, adenovirus, human T-leukemia virus I type, human T-leukemia virus II type, lymphocyte train of thought meningitis virus etc. (table 1).

Cause the design of primers (see table 2 nucleotide sequence) that encephalitis multiple gene detects, for RNA template, sequence is added: 5 '-GTACGACTCACTATAGGGA-3 ', adds sequence in PCR primer: 5 ,-AGGTGACACTATAGAATA-3 ' at 5 ' end of RT primer; For DNA profiling, add sequence at 5 ' end of arbitrary primer: 5 '-GTACGACTCACTATAGGGA-3 ', adds sequence at another primer: 5 '-AGGTGACACTATAGAATA-3 '.To adapt to GeXP reaction system.

The target site that table 1 detects

Table 2 nucleotide sequence

Concrete steps are as follows:

1. produce " causing encephalitis multiple gene detection kit ", test kit comprises: reverse transcription primer pipe, PCR primer pipe, reverse transcription buffer, PCR damping fluid, 25mM magnesium chloride, ThermoScript II, archaeal dna polymerase, fluorescent marker, positive control.

2. gather Patient Sample A's (blood, cerebrospinal fluid etc.), and extract nucleic acid

3. with patient's nucleic acid for template carries out reverse transcription (RT):

1) in 96 hole sample panel, reagent and sample (RT plate) is added in following ratio:

Note: positive control: 1 μ L/ reacts

2) hatch by following temperature after mixing:

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

4. be that template carries out PCR reaction with reverse transcription product

1) in 96 hole sample panel, reagent and sample (PCR plate) is added in following ratio:

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

2) thermal cycle reaction is carried out by following temperature after 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 PCR primer

5. electrocapillary phoresis sample separation

1) GeXP sample is prepared:

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

Note: PCR primer amount can be 0.1-1 μ L, or by loading after water on demand pre-dilution

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

3) electrocapillary phoresis sample separation.

6. interpretation of result.

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

Claims (1)

1. the multiple gene detection kit causing encephalitis based on electrocapillary phoresis, it is characterized in that comprising: reverse transcription primer pipe, PCR primer pipe, reverse transcription buffer, PCR damping fluid, 25mM magnesium chloride, ThermoScript II, archaeal dna polymerase, fluorescent marker, positive control, RT primer in described reverse transcription primer pipe, people RNA internal reference and reaction internal reference concrete nucleotide sequence as shown in the table, the PCR primer in described PCR primer pipe, people DNA internal reference and reaction internal reference concrete nucleotide sequence specifically as shown in the table:

The middle quoted passage contrast detecting target is as shown in the table:

Join in 96 hole sample panel and carry out reverse transcription, reaction conditions after the wherein nucleic acid samples RNA 5uL of RT reaction system and reaction conditions: 5-20ng/ul, DEPC water 8uL, 5 × RT damping fluid 4uL, RT primer solution 2uL, RT enzyme 1uL mixing: 48 DEG C 1 minute; 42 DEG C 60 minutes; 95 DEG C 5 minutes; 4 DEG C until collect RT product;

PCR reaction system and reaction conditions: RT product 9.3uL, the magnesium chloride 4uL of 5 × PCR damping fluid 4uL, 25mM, joins the reaction of 96 hole sample panel enterprising performing PCR, reaction conditions after PCR primer solution 2uL, archaeal dna polymerase 0.7uL mixing: 95 DEG C 10 minutes; 94 DEG C of 30 second, 60 DEG C of 30 second, 70 DEG C 1 minute, circulate 34 times; 70 DEG C 1 minute; 4 DEG C until collect PCR primer.