CN101113479A - Nucleic acid sequence of detecting rabies virus, detecting reagent case and detecting method - Google Patents

Abstract

The invention discloses a nucleotide sequence for detecting rabies virus, a detection kit and a method thereof, pertaining to inspection and quarantine field. A group of nucleotide sequence for detecting rabies virus comprises the nucleotide sequences shown in sequence tables from SEQ ID No. 1 to SEQ ID No. 3, wherein, the SEQ ID No. 1 and the SEQ ID No. 2 are respectively of a sense primer and an anti-sense primer while the SEQ ID No. 3 is of a fluorescence probe. The invention has the advantages of: (1) quickness, which shortens the detection period of 2-7 days in the traditional detection method to four hours; (2) sensitivity, the detection to mice brain virus of rabies virus CVS strain which is diluted by ten-time gradient shows that positive can be detected in object diluted to eight to ten times; (3) specificity, the detection to other collected pathogen from mated animals through established rabies virus fluorescence RT-PCR detection method shows a negative result and no cross reaction is found; (4) stability, repeated experimental results show the established method has good stability; (5) pollution resistance, a fully close reaction and no treatment after PCR ensure safe operation.

Description

Detect nucleotide sequence, detection kit and the detection method of rabies virus

Technical field

The present invention relates to detect nucleotide sequence, detection kit and the detection method of rabies virus, refer to a kind of fluorescent RT-PCR method for detecting fast and reagent thereof especially, belong to inspection and quarantine field.

Background technology

Rabies are high transmissible diseases of mortality ratio that a kind of people and all warm-blooded animals all can infect, and how to be bitten or to scratch and to be infected by carnivores such as the dog of carrying rabies virus, wolf, cat, mouse, also can be infected because of sucking infectious virus.Clinical manifestation be distinctive manic, frightened and restless, be afraid of that wind fears water, hydrostomia and pharyngismus, the threat to life to paralysis takes place eventually.Having infected animal that rabies virus do not fall ill can be used as the cause of disease reservoir host equally and propagates rabies.

China is one of human rabies district occurred frequently in the world, also is one of notebook disease country the earliest in the world.Human rabies is classified as Category B notifiable disease managed from country after the founding of the state, carry out the national dog motion of going out from nineteen fifty-one, the rabies sickness rate declines to a great extent, and the whole nation has only 5 provinces that case report is arranged, annual morbidity 40-463 example.Increase to some extent to the mid-1960s number of the infected, annual 103-1062 example, after the seventies, the rubies epidemiology scope is again and enlarges trend year by year: there was case in 14 provinces, the whole nation in 1971, developed into 25 provinces by 1988, annual report 4110-7028 example.Begin the eighties, because the Antirabic Vaccine applies, epidemic situation is descended year by year, but but ascendant trend appears in epidemic situation after the mid-90, national rabies morbidity number was 159 examples in 1996, rose to 230 examples, 234 example and 341 examples in 1997,1998,1999 3 years respectively, some do not have rabic area for many years and have occurred human rabies again.Calendar year 2001,2002 whole nation morbidity is respectively 891 example and 1122 examples, after the SARS epidemic situations in 2003, the Ministry of Health announces to society the epidemic situation of 27 kinds of legal serious infectious diseases that take place the first half of the year, what surprisingly, occupy the case fatality rate umber one remains an ancient disease---rabies.Still high until rabies epidemic situation in 2004,2005, several 2651 examples of whole nation report morbidity in 2004 were compared rising 30.14% with 2003, mortality ratio 100%, and the number of dying of illness occupies 37 kinds of Notifiable disease first places of China.Rabies death toll 2545 in 2005, death toll ranked second in various Notifiable diseases.According to the Ministry of Health in the recent period: the whole nation 21 provinces and regions in Jan-Sept, 2006, accumulative total is reported rabies several 2254 examples of falling ill, and increases by 29.69% over the same period last year, 2053 examples of dying in heaven.There are provinces and regions such as Guizhou, Guangxi, Hunan, Guangdong the more provinces and regions of falling ill.Rabies became the transmissible disease kind that causes China's death toll maximum in continuous 5 months.

Rabic control mainly depends on carries out immunization to the once local popular geographic dog of this disease, cat, to stop its infection; In addition, also to control the activity of dog, cat,, strengthen Isolation Quarantine and immunization external dog, cat in no rabies area.This old complaint is removed the cause of disease reservoir host that should comprise in the works in elimination and the minimizing wildlife.Many areas in the world, the main contagium of human rabies is dog and cat, and dog cat booster immunization inoculation and the quantity that reduces roam dog cat are remained the rabic effective measure of control.Even dog cat 100% immunity to artificial breeding, because differences such as vaccine quality, individual difference, inoculation means, the immunizing power that also can cause part dog cat not produce immunizing power or generation is not enough to resist the invasion and attack of rabies virus, moreover, can not block infection because of vaccinate in the saliva with the dog cat of poison.So the dog cat of process vaccine inoculation neither be perfectly safe, and how to guarantee the particularly safety of companion animals of dog cat, and then guarantee that the mankind avoid rabic invasion that key is vaccine inoculation and detection.Vaccine inoculation is to construct immunization barrier, and detection is the animal of finding to carry rabies virus early, and the measure of taking to slaughter reduces propagates risk, only in this way could be from the threat of control rabies in source to the people.And to the key issue of dog, the prevention and control of cat rabies--detect, to adopt detection technique fast and accurately exactly, reinforcement is to the monitoring of susceptible animal, reject infection animal, eliminate contagium, that is to say, crucial detection technique problem is to set up method responsive, that special being used to detects dog cat rabies virus and antibody, produce commercial detection reagent and apply, and particularly will be with malicious problem to be paid attention to the health of pet.

The fast method that is used for the rabies virus detection at present comprises that fluorescence antibody detects (FAT), methods such as immunohistochemical methods (IHC) and RT-PCR.But FAT and IHC are unsuitable for live body to be detected, and for some sample that receive in the laboratory, usually is outmoded sample, is unsuitable for histology and FAT and IHC.In addition, FAT and IHC are unsuitable for the detection of liquid sample, as saliva and celiolymph.

The RT-PCR method of common polymerase chain reaction not only can detect live virus nucleic acid, also can directly detect the nucleic acid fragment of deactivation, the sample wide material sources, and suitability is good.But the loaded down with trivial details relatively and easy pollution of this method operation, and what measure all is the end product of PCR, rather than the copy number of initiate dna or RNA.Owing to do not have linear relationship between the amount of the end product of PCR and the starting template amount,, can't accomplish accurately quantitative so can not calculate the copy number of initiate dna or RNA according to final PCR product amount.

Advantages such as the fluorescent quantitative PCR technique that emerges in recent years (also claiming the TaqMan technology) is highly sensitive with it, speed fast, high specificity are used widely at the aspects such as qualitative and detection by quantitative of gene expression dose analysis, pathogenic agent, and become the quantitative main method of current viral nucleic acid, it is quick, special, responsive and prevent the characteristics of crossed contamination more by force that fluorescent RT-PCR technology has, compare with common RT-PCR, sensitivity can improve about 100 times, can detect very micro-virus.We have applied it to the detection range of avian influenza virus, Avian pneumo-encephalitis virus, swine streptococcus and foot and mouth disease virus Asia 1 type, have obtained good technical effect.

The main points of TaqMan technology are to increase specific fluorescence double-tagging probe on the original a pair of Auele Specific Primer of regular-PCR basis.This probe joint position is positioned at the centre in PBR territory.5 ' end of probe and the different fluorescein of 3 ' end difference mark, fluorescein as 5 ' end mark, the fluorescence that it sends can receive by detected instrument, be called report fluorophor (representing) with R, the fluorescein of 3 ' end mark, in closely, can absorb the fluorescent signal that 5 ' end report fluorophor sends, be called cancellation fluorophor (representing) with Q.When PCR was reflected at annealing stage, primer and probe combined with target gene fragment simultaneously, and the fluorescent signal that the R group sends on this moment probe is absorbed by the Q group, and instrument detecting is less than fluorescent signal that R sent; The PCR reaction proceeds to extension during the stage, and the Taq enzyme is under the guiding of primer, along the synthetic new chain of template strand; When the extension of chain proceeds to the probe joint position, the function of its 5 ' → 3 ' exonuclease of Taq enzyme performance of this moment, probe is cut into mononucleotide, and the R group that meanwhile is marked on the probe dissociates out, and or else the fluorescence that R sent received by Q absorbs detected instrument.

PCR carries out a circulation, when having synthesized the new chain of N bar, with regard to hydrolysis N bar probe, also discharged the fluorophor of respective numbers.The amount of received fluorescence signal intensity of instrument and PCR reaction product is corresponding relation.Along with moving in circles of PCR reaction, the PCR product is exponential form and increases the also corresponding growth of fluorescent signal.If measured fluorescent value is an ordinate zou during with each PCR loop ends, be X-coordinate mapping with the PCR cycle number, can obtain a curve that connects each circulation back fluorescent value-be called amplification curve.When containing the nucleotide sequence that will detect pathogenic agent to some extent in detecting sample, resulting curve is " S " type; And in sample, do not contain pathogenic agent, and then the PCR process does not take place, and probe is not hydrolyzed, and does not produce fluorescent signal, and its amplification curve is a sea line.

The pcr amplification signal enters the lower limit of relatively stable increased logarithmic phase, is set in usually near the growth flex point place of S type amplification curve, is called threshold line (Threshold); And the cycle number in amplification curve and threshold line point of crossing is called the Ct value.The concentration of pathogenic agent is high more in the sample, and the Ct value is just more little.Measure pathogen nucleic acid in the key sample not with this method, can not only fast qualitative, also because advanced fluorescent signal detection system of fluorescent PCR itself and powerful information processing capability, can realize quantitative to pathogen nucleic acid.

The present invention is applied to fluorescent RT-PCR technology the rabies virus detection range first, and specific primer sequence, probe sequence, test kit and detection method at rabies virus are provided.

Technology contents

First purpose of the present invention provides the nucleotide sequence of strong, the highly sensitive detection rabies virus of a group-specific, comprises primer sequence and probe sequence.

Another object of the present invention provides the test kit of a kind of detection rabies virus quick, accurate, easy to use.

But the 3rd purpose of the present invention provides the method for a kind of quick, accurate detection by quantitative rabies virus.

For achieving the above object, the present invention is by the following technical solutions:

Select rabies virus non-coding region and N gene coding region particular sequence as target region, on the basis of multiple sequence comparison, carry out primer and probe design.Primer length is about 20 bases, and GC content is 50%-60%, no secondary structure and repeatability in the primer, and with the interior no complementary sequence of primer, the melting temperature (Tm) between primer (Tm value) differs less than 5 ℃ between primer.The length of probe is about 25 bases, and the Tm value is worth high about 5 ℃ than primer Tm.

One group of nucleotide sequence that detects rabies virus has the nucleotide sequence shown in sequence table SEQ ID No:1 to the SEQ ID No:3.

1)5’-AGT TGC AAA GCA AAA ATG TAA C-3′(SEQ ID NO：1)

2)5’-AGC AGG GTA CTT GTA CTC ATA TTG-3’(SEQ ID NO：2)

3)5’-[FAM]CCY CTA CAA TGG ATG CCG ACA RGA TTG[TAMRA]-3’(SEQ IDNO：3)

Wherein sequence 1) and 2) be respectively sense primer and antisense primer, sequence 3) for annexing fluorescent probe, 5 ' end mark report fluorophor FAM (6-Fluoresceincarboxylic acid) of probe, 3 ' end mark cancellation fluorophor TAMRA.

We adopt TaqMan fluorescent PCR detection technique to set up the rabies virus detection method, the various conditions of reaction are optimized, comprising the screening of primer probe, Mg ²⁺The optimization of working concentration, the optimization of primer concentration and probe concentration obtains following test kit.

A kind of test kit that detects rabies virus, the 48tests/ box, composed of the following components:

1) lysate: available from INVITROGEN company, carry out packing, 6 pipe/boxes ,-20 ℃ of preservations by the 5ml/ pipe;

2) RT-PCR reaction solution, see the following form:

Table 1

Component	Final concentration
		10 * PCR damping fluid	0.5 * PCR damping fluid
25mM MgCl 2	4.0mM
		2.0mM dNTP	0.2mM dNTP
Primer
	1	0.2μmol/L
Primer
	2	0.2μmol/L
Probe			0.1μmol/L

10 * PCR damping fluid, 25mM MgCl ₂, 2.0mM dNTP all purchases the company in Promega; Primer and probe all entrust Dalian precious biotinylated biomolecule Engineering Co., Ltd synthetic, wherein consisting of of 10 * PCR damping fluid: 500mMKCl, 100mM Tris-HCl (25 ℃ of pH9.0), 1.0%Triton X-100.

3) RT-PCR enzyme granulate, available from AMERCIA company, 1/pipe * 12 pipes; 12 pipe/boxes.

4) Taq archaeal dna polymerase 5U/ μ L, 15 μ L * 1 pipe are available from Promega company.

5) DEPC water, 1mL * 1 pipe; With twice distillation of tap water, through Millipore MILLI-Q PF PLUS pure water instrument purifying, collect the water of resistivity 〉=18.0M Ω .cm, the Millipore-Q purified water add DEPC to final concentration be 0.1%, 37 ℃ of stir process 12hr, 15lbf/in2 (1.034 * 105Pa) high pressure steam sterilizations 15 minutes.

6) negative control: 1mL * 1 pipe, the aseptic SPF mouse substantial viscera of winning is made 20% suspension with 0.01mol/L pH7.2 PBS buffer saline, and 70 ℃ act on 1 hour.

7) positive control: the non-infectious RNA fragment for in-vitro transcription has the nucleotide sequence shown in the sequence table SEQ ID No.4.

127 bp purpose fragment gene sequences (SEQ ID No.4) are as follows:

1 AGTTGCAAAG CAAAAATGTA ACACCTCTAC AATGGATGCC GACAGGATTG TATTCAGAGC

61 TAATAATCAG GTGGTCTCTT TGAGGCCTGA GATTATCGCT GATCAATATG AGTACAAGTA

121 CCCTGCT

The preparation method of positive control is as follows: the RT-PCR amplified production that reclaims rabies virus, obtain the highly purified fragment that contains the 127bp of rabies virus part leader sequence and part N gene, (available from Promega company) is connected with PGEM-T easy carrier, transform recombination bacillus coli JM109 competent cell, alkaline lysis method of extracting plasmid DNA, cut through PCR and enzyme and to identify the back positive recombinant plasmid of acquisition as positive control, called after PGEM-RAB.Plasmid with purifying is a template, carries out in-vitro transcription with the Ribo MAXTM Large Scale RNA ProductionSystem-T7 test kit of Promega company; After the in-vitro transcription product removed wherein dna profiling with DNase, promptly obtain preparing the required RNA positive reference substance mother liquor of AIV positive reference substance.Present method is this area ordinary method, but this positive control sequence synthetic or the plasmid preparation that obtains with other similar approach.

Synthetic primer and probe are HPLC analyze, as the ratio that obtains single absorption peak collection of illustrative plates and measure OD260nm/OD280nm with ultraviolet spectrophotometer promptly is considered as qualified primer between 1.6-2.0.The RNA that extracts is that template increases from the rabies virus culture of dilution in 1: 100, and the result shows that primer provided by the invention pair is used with probe, and the Ct value of amplification is relatively low, and increasing degree is higher.

The primer concentration that screening is good is that spacing increases progressively from 0.1 μ M to 0.8 μ M with 0.1 μ M, and concentration and probe concentration increases progressively with 0.025 μ M from 0.025 μ M to 0.2 μ M.Proportioning to primer and probe different concns compares, from repeatedly finding the revision test: the primer of different concns, probe are for this positive template, the CT value is basicly stable about 18.55, but primer concentration is 0.2 μ M, fluorescence amplification was higher relatively when concentration and probe concentration was 0.1 μ M, so selected primer concentration is 0.2 μ M, concentration and probe concentration is primer and the concentration and probe concentration of 0.1 μ M as the rabies virus fluorescent RT-PCR method for detecting.

We are at Roche Light Cycler and ABI 7900HT fluorescent PCR detector, at 42 ℃/30min, and 92 ℃/3min; And 92 ℃/10sec, 45 ℃/30sec, after the reverse transcription of 72 ℃/60sec (5 circulations) and the pre-amplification program, in this experiment denaturation temperature and time, annealing and elongating temperature and time having been carried out optimization experiment.During amplification, adopt cycle index to be respectively 40,45 and 50, the relatively Ct value of amplification, and then the cycle index of definite amplification.Final definite expanding effect that adopts is better, and short scheme consuming time is the PCR reaction parameter: 92 ℃/5sec, 55 ℃/10sec, fluorescence is collected in 40 circulations of 72 ℃/20sec during each loop ends.

Studies show that Mg ²⁺Concentration is bigger to the influence of fluorescent PCR amplification, thus use the good primer probe of screening, with Mg ²⁺Concentration be that spacing increases progressively with 0.5mM from 1.5mM to 6.0mM, to different Mg ²⁺Amplification under the concentration conditions compares, and the result shows Mg ²⁺Concentration is relevant to the sensitivity of fluorescence increment and detection, improves its working concentration, can improve the fluorescence increment of detection probes, but along with Mg ²⁺The raising of concentration when particularly surpassing 5mM, when detecting some sample, can be found the phenomenon of floating on the curve.For the rabies virus detection method, the Mg after the optimization ²⁺Concentration is 4.0mM.

The Taq enzyme that we select Promega company to produce, the definition of a unit: 74 ℃ act on 30 minutes, the dNTPs of 10nM can be mixed needed enzyme amount in the acid soluble material.The active requirement: tool dna polymerase activity and 5 ' → 3 ' exonuclease activity, do not have 3 ' → 5 ' exonuclease activity and endonuclease activity; The tool thermostability, 94 ℃ still kept 50% activity after 1 hour.The optimization experiment template of Taq enzyme dosage (in the U of unit) increases after adopting rabies virus culture (dilution in 1: 100) to extract the RNA reverse transcription.From repeatedly selecting 1.25U Taq enzyme as the Taq enzyme amount of using the revision test result.

A kind of method that detects rabies virus comprises the steps:

1) extracts sample RNA;

2) the sample RNA that extracts is carried out the RT-PCR amplification:

Primer sequence is 1) 5 '-AGT TGC AAA GCA AAA ATG TAA C-3 ' (SEQ ID NO:1)

2)5’-AGC AGG GTA CTT GTA CTC ATA TTG-3’(SEQ ID NO：2)

Fluorescent probe is 3) 5 '-[FAM] CCY CTA CAA TGG ATG CCG ACA RGA TTG

[TAMRA]-3’(SEQ ID NO：3)

Amplification condition is 42 ℃/30min, 92 ℃/3min; 92 ℃/10sec, 45 ℃/30sec, 72 ℃/60sec, 5 circulations; 92 ℃/5sec, 55 ℃/10sec, fluorescence is collected in 40 circulations of 72 ℃/20sec during each loop ends;

3) whether the fluorescence intensity of mensuration RT-PCR reaction system exists rabies virus in the judgement sample; Feminine gender, no Ct value and do not have amplification curve, showing does not have rabies virus in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is rabies virus in the expression sample.

Described step 1) needs sample is handled before extracting sample RNA, method is: for tissue sample, fully grind in mortar with aseptic scissors and tweezers clip sample 1.0g to be checked, add 5mL PBS mixing again, then tissue suspension is changed in the aseptic Eppendorf pipe, number standby.For liquid sample, directly draw to aseptic Eppendorf pipe with asepsis injector, number standby.The sample of gathering or handling is preserved under 2 ℃～8 ℃ conditions and should be no more than 24h; If need prolonged preservation, must place-70 ℃ of refrigerators, but should avoid multigelation (freeze thawing is 3 times at most).After the sample sealing of gathering, adopt thermo jug or insulated tank sealing on the rocks, be transported to the laboratory as early as possible.

The method that described step 1) is extracted sample RNA is (carrying out in the sample process district): get n 1.5ml sterilization centrifuge tube (n=sample number+1 pipe negative control+1 pipe positive control), perform mark.(lysate has very strong corrodibility at first to add the 600ul lysate, be sure not to be stained with skin or clothing, otherwise immediately with a large amount of flushing with clean water and dry), add sample to be tested, negative control, each 200ul of positive control (a sample is used a suction nozzle instead) then respectively; Add the 200ul chloroform again, vibration mixing 5sec (should not be too strong, in order to avoid produce emulsion layer, also available hand is put upside down mixing) on the vortex mixer.

12, the centrifugal 15min of 000g (as have ready conditions, carry out centrifugal in 4 ℃).Get with the last step in the 1.5ml sterilization centrifuge tube of equal amts, add 400ul Virahol (20 ℃ of precoolings), perform mark.Draw in the extremely corresponding pipe of supernatant liquor phase transition in above-mentioned each pipe, put upside down mixing.

12, the centrifugal 15min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids (different samples should be stained with dried in the different places of thieving paper); Add 600ul 75% ethanol (20 ℃ of precoolings), put upside down washing.

12, the centrifugal 10min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids as far as possible.4, the centrifugal 10sec of 000g is thrown to the pipe bottom with the residual liquid on the tube wall, it is blotted with micro sample adding appliance that (a sample is used a suction nozzle instead as far as possible, the attention suction nozzle is not run into the precipitation one side), drying at room temperature 3min (should not be too dry) in order to avoid RNA is insoluble.

Add 11ul DEPC water, mixing dissolves the RNA on the tube wall gently, and 2, the centrifugal 5sec of 000g preserves standby (note: the RNA of this moment is subject to the RNA enzyme liberating most, please carries out pcr amplification in 2 hours) on ice.

Described step 2) the sample RNA that extracts being carried out RT-PCR amplification concrete operations is: from test kit, take out RT-PCR reaction solution, Taq enzyme, and after at room temperature melting, 2, the centrifugal 5sec of 000g.If required PCR pipe number is n (a n=sample number+1 pipe negative control+1 pipe positive control), each test reaction system needs 15ul RT-PCR reaction solution and 0.25ulTaq enzyme.Calculate the usage quantity of good each reagent, add in the proper volume test tube, to wherein adding/go into n/4 RT-PCR enzyme granulate, thorough mixing is even, and each packing 15ul in each PCR pipe is transferred to the sample process district.Each 10ul of RNA solution that adds preparation in the PCR of each setting pipe respectively, the tight pipe lid of lid is in the centrifugal 5sec of 400g.The PCR pipe sequenced put into the fluorescent PCR detector, the record sample is put order.

Reaction parameter is provided with:

---fs, pre-42 ℃/30min of sex change, 92 ℃/3min;

---subordinate phase, 92 ℃/10sec, 5 circulations of 72 ℃/1min of 45 ℃/30sec;

---phase III, 92 ℃/5s, 55 ℃/10s, 40 circulations of 72 ℃/20sec, phosphor collection setting

Carry out when each round-robin annealing is extended in the phase III.

Described step 3) is measured the fluorescence intensity of RT-PCR reaction system, and whether exist the method for rabies virus to be in the judgement sample: the threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve.Different instruments can be adjusted according to instrument noise situation.Quality control standard, negative control do not have the Ct value and do not have amplification curve.The Ct value of positive control answers≤30.0, and specific amplification curve occurs.As negative control and the discontented condition that is enough to of positive condition, it is invalid that experiment this time is considered as.The result describes and judges, feminine gender, and no Ct value and do not have amplification curve, showing does not have rabies virus in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is rabies virus in the expression sample.

Advantage of the present invention is: the present invention selects rabies virus non-coding region and N gene coding region as target region, design primer and probe foundation have also been optimized the rabies virus fluorescent RT-PCR method for detecting, obtained excellent technique effect, be not only applicable to accurate detection to rabies virus in the humans and animals tissue samples, also applicable to humans and animals biopsy sample (skin living tissue, saliva and celiolymph) detection, can be widely used in the examination of domestic companion animals rabies virus, the laboratory rapid detection of quarantine of entry and exit companion animals and human rabies:

1) quick: this method is monitored in real time to the PCR product, and RT-PCR finishes to obtain the result, and 2～7 day detection time of traditional detection method shortened to 4 hours.

2) sensitivity: owing to adopted specific fluorescent probe and highly sensitive fluorescent PCR instrument, make this method highly sensitive 100～1000 times than traditional PCR method; With the method for being set up the rabies virus CVS strain mouse brain poison with 10 times of gradient dilutions is detected, the result shows and is diluted to 10 ^-8Doubly all detect positive.

3) special: owing to not only adopted specific primer, and adopted specific probe, make the specificity of this method be higher than traditional RT-PCR method; The rabies virus fluorescent RT-PCR method for detecting of setting up is detecting other collected companion animals cause of diseases, and the result is negative, does not find cross reaction;

4) stable: the replica test result shows having good stability of institute's establishment method;

5) be difficult for polluting: totally-enclosed reaction need not the PCR aftertreatment, operational safety.

The invention will be further described below in conjunction with specification drawings and specific embodiments, do not limit the present invention in any way.Every any this area of carrying out according to the disclosure of invention is equal to replacement, all belongs to protection scope of the present invention.

Description of drawings

Fig. 1 is the limit of detection measurement result of rabies virus fluorescent RT-PCR method for detecting.

Fig. 2 is for adopting the limit of detection result of viral separation and combination fluorescence antibody detection technique.

Fig. 3 is for carrying out the result of relative quantification to viral RNA.

Fig. 4 is the specificity test-results of rabies virus fluorescent RT-PCR method for detecting.

Fig. 5 is the detected result of rabies virus fluorescence RT-PCR method to artificial challenge SPF mouse tissue internal organs.

Fig. 6 is the detected result of the common RT-PCR method of rabies virus to artificial challenge SPF mouse tissue internal organs.

Fig. 7 adopts the detected result of fluorescence RT-PCR to commercialized vaccine and clinical sample (1～26).

Fig. 8 adopts the detected result of fluorescence RT-PCR to clinical sample (27～64).

Embodiment

Embodiment 1: the preparation of test kit and use

1. the preparation of test kit is formed, and sees Table 2.

Table 2: the test kit preparation is formed

Form by (48 tests/ box)	Quantity
		Lysate	5.0mL * 6 pipes
Rabies virus fluorescence RT-PCR reaction solution	750 μ L * 1 pipe
		Taq enzyme (5U/ μ L)	15 μ L * 1 pipe
The RT-PCR enzyme granulate	1 * 12 pipes
		DEPC water	1mL * 1 pipe
Negative control	1mL * 1 pipe
		Positive control	1mL * 1 pipe

2. the using method of test kit

1) DNA extraction:

Get n 1.5ml sterilization centrifuge tube (n=sample number+1 pipe negative control+1 pipe positive control), perform mark.(lysate has very strong corrodibility at first to add the 600ul lysate, be sure not to be stained with skin or clothing, otherwise immediately with a large amount of flushing with clean water and dry), add sample to be tested, negative control, each 200ul of positive control (a sample is used a suction nozzle instead) then respectively; Add the 200ul chloroform again, vibration mixing 5sec (should not be too strong, in order to avoid produce emulsion layer, also available hand is put upside down mixing) on the vortex mixer.

12, the centrifugal 15min of 000g (as have ready conditions, carry out centrifugal in 4 ℃).Get with the last step in the 1.5ml sterilization centrifuge tube of equal amts, add 400ul Virahol (20 ℃ of precoolings), perform mark.Draw in the extremely corresponding pipe of supernatant liquor phase transition in each pipe, put upside down mixing.

12, the centrifugal 15min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids (different samples should be stained with dried in the different places of thieving paper); Add 600ul 75% ethanol (20 ℃ of precoolings), put upside down washing.

12, the centrifugal 10min of 000g.Remove supernatant gently, be inverted on the thieving paper, be stained with dry liquids as far as possible.4, the centrifugal 10sec of 000g is thrown to the pipe bottom with the residual liquid on the tube wall, it is blotted with micro sample adding appliance that (a sample is used a suction nozzle instead as far as possible, the attention suction nozzle is not run into the precipitation one side), drying at room temperature 3min (should not be too dry) in order to avoid RNA is insoluble.

Add 11ul DEPC water, mixing dissolves the RNA on the tube wall gently, and 2, the centrifugal 5sec of 000g preserves standby (note: the RNA of this moment is subject to the RNA enzyme liberating most, please carries out pcr amplification in 2 hours) on ice.

2) amplifing reagent is prepared and preparation

From test kit, take out RT-PCR reaction solution, Taq enzyme, after at room temperature melting, 2, the centrifugal 5sec of 000g.If required PCR pipe number is n (a n=sample number+1 pipe negative control+1 pipe positive control), each test reaction system needs 15ul RT-PCR reaction solution and 0.25ul Taq enzyme.Calculate the usage quantity of good each reagent, add in the proper volume test tube, to wherein adding n/4 RT-PCR enzyme granulate, thorough mixing is even, and each packing 15ul in each PCR pipe is transferred to the sample process district.Each 10ul of RNA solution that adds preparation in the PCR of each setting pipe respectively, the tight pipe lid of lid is in the centrifugal 5sec of 400g.The PCR pipe sequenced put into the fluorescent PCR detector, the record sample is put order.

Reaction parameter is provided with:

---fs, pre-42 ℃/30min of sex change, 92 ℃/3 min;

---subordinate phase, 92 ℃/10sec, 5 circulations of 72 ℃/1min of 45 ℃/30sec

---phase III, 92 ℃/5s, 55 ℃/10s, 40 circulations of 72 ℃/20sec, phosphor collection

Be arranged on when each round-robin of phase III is annealed extension and carry out.

3) result judges

The interpretation of result condition enactment reads detected result.The threshold setting principle is with the vertex of threshold line just above normal negative control product amplification curve.Different instruments can be adjusted according to instrument noise situation.Quality control standard, negative control do not have the Ct value and do not have amplification curve.The Ct value of positive control answers≤30.0, and specific amplification curve occurs.As negative control and the discontented condition that is enough to of positive condition, it is invalid that experiment this time is considered as.The result describes and judges, feminine gender, and no Ct value and do not have amplification curve, showing does not have rabies virus in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is rabies virus in the expression sample.

Embodiment 2: the sensitivity test of test kit and be used for the relative quantification of viral RNA

1. material:

The classic rabies virus type strain CVS (6.33lgLD50/0.03ml) that the virus strain that is applied in the method research process is preserved for this laboratory.

2. method

1) type strain CVS strain rabies virus mouse brain poison is done 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8, 10 ^-9, 10 ^-10Doubly dilution, carrying out the rabies virus fluorescence RT-PCR detects, simultaneously with each dilution mouse brain poison inoculation BHK21 clone, cultivate after 72 hours for 37 ℃, with the dehydrated alcohol of precooling spend the night fixing after, dye 37 ℃ of 2h that dye with the rabies fluorescence antibody, take pictures with the inverted fluorescence microscope observation, relatively the sensitivity of two kinds of methods.

2) relative quantification of viral RNA:

For realizing the relative quantification to viral RNA amount in the clinical sample, we further detect the viral RNA sample of continuous 10 times of dilutions, and the amount of viral RNA in Ct value and the sample is carried out linear regression, calculate relation conefficient.

3. result

1) see illustrated in figures 1 and 2, the rabies virus culture of deactivation is made 10 times of serial dilutions, condition after utilization is optimized detects with two kinds of detection methods respectively, and test-results shows that the limit of detection of the detection method of foundation can reach 10 for type strain CVS strain mouse brain poison ^-8, Fig. 2 is the sensitivity detected result of rabies virus detection method.And adopt BHK21 clone, and cultivate after 72 hours for 37 ℃, carry out fluorescent antibody staining, sensitivity only can reach 10 ^-5

2) relative quantification of viral RNA: for realizing the relative quantification to viral RNA amount in the clinical sample, we further detect the viral RNA sample of continuous 10 times of dilutions, and the amount of viral RNA in Ct value and the sample is carried out linear regression, calculate relation conefficient.The result shows that the amount of viral RNA is linear dependence with the Ct value that test records, and R2=0.99111193 shows that the method for foundation can be used for the definite relatively of viral RNA amount, as shown in Figure 3.

Embodiment 3: the specificity test of test kit

1. material

Table 3: the virus strain that is applied in the method research process

Virus	The source
		Dog I type adenovirus canine parvovirus canine distemper virus	This laboratory is preserved this laboratory and is preserved this laboratory preservation

2. method

With the method for being set up the common various viruses of dog (comprising canine distemper virus, canine parvovirus, dog I type adenovirus) are detected, with the specificity of verification method.

3. result

As shown in Figure 4, use the method for being set up that the various viruses of common dog (comprising canine distemper virus, canine parvovirus, dog I type adenovirus) are detected, the result shows method and the above-mentioned viral no cross reaction of being set up, and specificity is good.

Embodiment 4: test kit is to the detection test of artificial challenge's mouse tissue internal organs

1. material:

The classic rabies virus type strain CVS (6.33lgLD50/0.03ml) that the virus strain that is applied in the method research process is preserved for this laboratory.

2. method

In 3 grades of laboratories of Biosafety, to 10 mouse, behind intracranial inoculation rabies virus type strain (6.33lgLD50/0.03ml), the result shows that mouse began morbidity in back 5 days in inoculation, shows as the imbalance of fortune step, muscular tremor, paralysis at last.Infect after 10 days, the artificial dead mouse that causes death is asepticly won the heart, liver, spleen, and lung, kidney, brain, eye, skin is made 20% suspension, gets 200 μ l and extracts RNA, detects with the test kit of developing and the common RT-PCR of bibliographical information respectively.

3. result

As shown in Figure 5 and Figure 6, the result shows that all samples is all positive, and different tissues toxic amount difference when adopting fluorescence RT-PCR to detect, and each organizes toxic amount putting in order from high to low to be brain, eye, lung, kidney, heart and skin, liver and spleen; For same sample, adopt RT-PCR to detect, the heart, liver, spleen, lung, brain, kidney, eye is positive, and skin is negative.

Embodiment 5: rabies virus fluorescence RT-PCR test kit batch between, batch in repeatable test

For between this test kit is criticized, batch in repeatability comprehensively examine, we select three batches of qualified reagent carried out batch between, batch in repeatable test.

1. experiment material

Reagent: 3 batches of rabies virus fluorescence RT-PCR test kits, lot number: 200612001,200612002,200612003.

Detecting instrument: full-automatic fluorescent PCR detector ROCHE company product, model, LightCyclerII.

Detect sample: rabies virus CVS strain mouse brain poison is done 10 ^-3, 10 ^-4, 10 ^-5Doubly after the dilution, called after D1, D2, D3 respectively are between continuous criticize with the rabies virus fluorescent RT-PCR method for detecting respectively for three times, criticize interior replica test.

2. experiment content: the every batch of reagent all carries out 3 fluorescence RT-PCRs to 3 extent of dilution of rabies virus CVS strain mouse brain poison and detects, 10 parts of multiple pipes of each pattern detection at every turn, then according to above data computation test kit batch in, batch between error.

Detected result requires: same sample batch in, batch between CV value≤10%

3. experimental result

The repeatability of each 10 parts in each sample being answered pipes according to " the rabies virus fluorescent RT-PCR detection reagent box " of visible 200612001,200612002,200,612,003 3 lot numbers of table 4 result detects, same lot number, with the CV value of a detected result all less than 10%, illustrate that this test kit detects simultaneously and has good repeatability with criticizing reagent.

Table 4: the three batches of test kits criticize interior, batch between the replica test data

Lot number	Number of times	Sample	Each detected result (Ct value)										Homogeneous detection statistics result in crowd
																1	2	3	4	5	6	7	8	9	10	Average	Standard deviation	The CV value
			200612001 batches of test kits	1	D1	12.74	12.61	13.01	12.88	12.77	12.54	12.67	12.89	13.05	12.45														12.76	0.198	1.55％
D2	16.55	16.43														16.32	16.20	16.98	16.44	16.77	16.99	17.05	16.75	16.65	0.302	1.82％
					D3	20.51	20.40	20.88	21.09	20.48	20.69	20.91	21.16	20.72	20.38												20.72	0.281	1.36％
2	D1	12.33														12.81	12.95	12.77	12.47	13.04	13.17	12.88	13.01	12.65	12.81	0.263				2.05％
				D2	16.32	16.44	16.78	16.19	16.98	16.56	16.27	16.37	17.01	17.02	16.59												0.326	1.96％
	D3	20.44														20.90	20.78	20.99	20.67	20.34	20.61	21.07	20.52	20.77	20.71	0.238			1.15％
				3	D1	12.13	12.58	12.75	12.66	12.23	12.94	12.85	12.01	12.91	12.88												12.59	0.347		2.76％
D2	16.71	16.22														16.43	16.09	16.58	16.57	16.80	16.91	17.02	16.82	16.62	0.299	1.80％
					D3	20.80	20.65	20.47	20.39	20.88	20.79	20.19	20.37	20.60	20.70												20.58	0.223	1.08％
200612002 batches of test kits	1	D1														12.55	12.61	12.75	12.20	12.60	12.51	12.80	12.79	12.66	12.63	12.61				0.174	1.38％
			D2	16.88	16.99	16.76	16.83	16.85	16.32	16.72	16.73	16.88	16.22	16.72	0.251												1.50％
		D3														20.40	20.71	20.83	20.90	20.57	20.38	20.77	20.48	20.84	20.83	20.67		0.197	0.95％
			2	D1	12.61	12.37	12.87	12.34	12.60	12.59	12.11	12.09	13.06	12.99	12.56												0.341			2.71％
	D2	16.91														16.77	16.46	16.33	16.78	16.32	16.62	16.48	16.96	16.44	16.61	0.235		1.42％
				D3	20.77	20.96	20.33	20.49	20.37	20.31	20.66	20.98	20.87	20.64	20.64												0.256		1.24％
	3	D1														12.53	12.54	12.67	12.67	12.39	12.40	12.51	12.78	13.03	13.09	12.66		0.243		1.92％

		D2	16.74	16.78	16.57	16.39	16.78	16.42	16.62	16.67	16.76	16.90	16.66	0.164	0.99％
																D3	20.78	20.36	20.48	20.60	20.70	20.77	20.61	20.99	20.77	20.94	20.7	0.194	0.94％
		200612003 batches of test kits	1	D1	12.54	12.53	12.57	12.84	12.79	12.48	12.31	12.75	12.40	12.88	12.61															0.195	1.55％
D2	16.89															16.68	16.20	16.37	16.52	16.45	16.61	16.77	16.80	16.70	16.60	0.214	1.29％
				D3	21.05	20.56	20.29	20.77	20.68	20.39	20.66	20.78	20.54	20.55	20.63													0.215	1.04％
2	D1															12.99	12.97	12.91	12.88	12.28	12.38	12.40	12.45	12.91	12.71	12.69	0.280			2.21％
			D2	16.81	16.87	16.30	16.40	16.88	16.49	16.66	16.69	16.89	16.91	16.69	0.223													1.34％
	D3															20.66	20.48	20.66	20.49	20.55	20.56	20.24	20.59	20.77	20.94	20.59	0.186		0.90％
			3	D1	12.56	12.67	12.77	12.35	12.62	12.49	12.19	12.89	12.56	12.47	12.56													0.201		1.60％
D2	16.91															16.76	16.46	16.58	16.39	16.60	16.72	16.39	16.66	16.79	16.63	0.176	1.06％
				D3	20.80	20.66	20.71	20.40	20.76	20.87	20.40	20.67	20.30	20.81	20.64													0.199	0.97％

" rabies virus fluorescent RT-PCR detection reagent box " according to visible 200612001,200612002,200,612,003 3 lot numbers of table 5 result detects three increments repeatability originally, same lot number, the CV value between three detected results illustrates that all less than 10% this test kit also has good repeatability with the detected result of batch reagent, different time separately.

Table 5: replica test data in three batches of test kits are criticized separately

The reagent lot number	Detect sample	Detect sample number	Repeated statistics in batch
						Average Ct value	Standard deviation	The CV value
			200612001	D1	30				12.72	0.269	2.11％
D2
		30		16.62	0.309	1.86％
D3
		30	20.67	0.247	1.19％
200612002						D1	30	12.61	0.253	2.00％
	D2
			30	16.66	0.217	1.30％
	D3
		30	20.67	0.216	1.04％
	200612003					D1	30	12.62	0.225	1.79％
D2
			30	16.64	0.204	1.23％
D3
		30	20.62	0.200	0.97％

" rabies virus fluorescent RT-PCR detection reagent box " according to visible 200612001,200612002,200,612,003 3 lot numbers of table 6 result detects three increments repeatability originally, three lot number reagent, the CV value between three detected results illustrates that all less than 10% the detected result of this test kit different batches reagent, different time also has good repeatability separately.

Table 6: three batches of test kits are criticized a replica test data

The reagent lot number	Detect sample	Detect sample number	Repeated statistics between batch
						Average Ct value	Standard deviation	The CV value
			200612001 200612002 200612003	D1	90				12.65	0.249	1.97％
D2	90	16.64				0.243	1.46％
				D3	90			20.65	0.221	1.07％

According to above testing data and statistical study, that this test kit is criticized is interior, batch between repeated result's CV value all less than 10%, illustrate have well batch in, repeated between criticizing.

Embodiment 6: to the laboratory report of clinical sample and commercialized vaccine detection

1. material

64 parts of dog saliva samples that pet hospital, Beijing provides; And four kinds of commercialized vaccines (the rich road rabies inactivated vaccine RabvacTM3 of the U.S., special 5 attenuated live vaccines of hundred departments of the national Ministry of Agriculture, Intervet Nobivac dog cat rabies inactivated vaccine and Rabisin-R Rui Beikang Rabies Vaccine).

2. method

For these 64 parts of clinical samples, be divided into 2 parts, 1 part is adopted fluorescent RT-PCR method for detecting to detect, the viral separation and combination fluorescent antibody staining of another part employing detects and (adopts the BHK-21 monolayer cell to carry out the virus separation, adopt fluorescent antibody staining behind the inoculation 72h), the detected result of two kinds of methods of comparison.For commercialized vaccine, directly extract RNA and carry out the fluorescence RT-PCR detection.

3. result

Detected result sees Table 7, Fig. 7 and Fig. 8.

In this research, we tentatively are used for the detection of clinical sample with the method for setting up, and sample is 64 parts of dog saliva samples that pet hospital, Beijing provides, and the result shows with viral separation and combination fluorescent antibody test result in full accord.Detected result to four kinds of commercialized vaccines shows, the rich road rabies inactivated vaccine RabvacTM3 of the U.S., the Ministry of Agriculture of country hundred special 5 attenuated live vaccines of department and Intervet Nobivac dog cat rabies inactivated vaccine are positive, and the detected result of Rabisin-R Rui Beikang Rabies Vaccine is negative.

The detected result of table 7:64 part clinical sample

Sample	Umber	The fluorescence RT-PCR detected result	Virus separation detection qualification result
				The asymptomatic dog saliva of the positive dog saliva of the positive dog saliva of CDV CPV	9 5 50	0/9 0/5 0/50	0/9 0/5 0/50

Sequence table

＜110〉People's Republic of China Beijing Entry-Exit Inspection and Quarantine Bureau

＜120〉nucleotide sequence, detection kit and the detection method of detection rabies virus

<130>

<160>4

<170>PatentIn version 3.3

<210>1

<211>22

<212>DNA

＜213〉synthetic

<400>1

agttgcaaag caaaaatgta ac 22

<210>2

<211>24

<212>DNA

＜213〉synthetic

<400>2

agcagggtac ttgtactcat attg 24

<210>3

<211>27

<212>DNA

＜213〉synthetic

<400>3

ccyctacaat ggatgccgac argattg 27

<210>4

<211>127

<212>DNA

＜213〉synthetic

<400>4

agttgcaaag caaaaatgta acacctctac aatggatgcc gacaggattg tattcagagc 60

taataatcag gtggtctctt tgaggcctga gattatcgct gatcaatatg agtacaagta 120

ccctgct 127

Claims (6)

1. one group of nucleotide sequence that detects rabies virus, has the nucleotide sequence shown in sequence table SEQ ID No:1 to the SEQ ID No:3, wherein sequence SEQ ID No:1 and SEQ ID No:2 are respectively sense primer and antisense primer, and sequence SEQ ID No:3 is a fluorescent probe.

2. the nucleotide sequence of detection rabies virus according to claim 1 is characterized in that: 5 ' the end mark report fluorophor FAM of SEQ ID No:3,3 ' end mark cancellation fluorophor TAMRA.

3. test kit that detects rabies virus, 48 tests/ boxes, composed of the following components:

1) lysate: 5ml/ pipe, 6 pipe/boxes ,-20 ℃ of preservations;

2) RT-PCR reaction solution, 750 μ L * 1 pipe comprise: 10 * PCR damping fluid, 25mM MgCL ₂, 2.0mM dNTP, primer 1, primer 2 and probe;

3) RT-PCR enzyme granulate, 1/pipe * 12 pipes;

4) Taq archaeal dna polymerase 5U/ μ L, 15 μ L * 1 pipe;

5) DEPC water, 1mL * 1 pipe;

6) negative control: 1mL * 1 pipe: the aseptic SPF mouse substantial viscera of winning, make 20% suspension with 0.01mol/L pH7.2 PBS buffer saline, 70 ℃ act on 1 hour;

7) positive control: the non-infectious RNA fragment for in-vitro transcription has the nucleotide sequence shown in the sequence table SEQ ID No:4.

4. a kind of test kit that detects rabies virus according to claim 3 is characterized in that: described primer sequence is the nucleotide sequence shown in sequence table SEQ ID No:1 and the SEQ ID No:2.

5. a kind of test kit that detects rabies virus according to claim 3, it is characterized in that: described probe sequence is the nucleotide sequence shown in the sequence table SEQ ID No:3, its 5 ' end mark report fluorophor FAM, 3 ' end mark cancellation fluorophor TAMRA.

6. a method that detects rabies virus comprises the steps:

1) extracts sample RNA;

2) the sample RNA that extracts is carried out the RT-PCR amplification:

Primer sequence is 1) 5 '-AGT TGC AAA GCAAAAATG TAA C-3 '

2)5’-AGC AGG GTA CTT GTA CTC ATATTG-3’

Fluorescent probe is 5 '-[FAM] CCY CTA CAA TGG ATG CCG ACA RGA TTG[TAMRA]-3 '

Amplification condition is 42 ℃/30min, 92 ℃/3min; 92 ℃/10sec, 45 ℃/30sec, 72 ℃/60sec, 5 circulations; 92 ℃/5sec, 55 ℃/10sec, fluorescence is collected in 40 circulations of 72 ℃/20sec during each loop ends;

3) whether the fluorescence intensity of mensuration RT-PCR reaction system exists rabies virus in the judgement sample; Feminine gender, no Ct value and do not have amplification curve, showing does not have rabies virus in the sample; The positive, Ct value≤30.0, and specific amplification curve appears, there is rabies virus in the expression sample.

Images