CN209636254U - A kind of kit for cerebrospinal fluid difficulty pathogen diagnosis - Google Patents

Abstract

This application discloses a kind of kits for cerebrospinal fluid difficulty pathogen diagnosis, not only include reagent box body, further include fixed plate, primer plate and 96 hole PCR reaction plates positioned at reagent box body；It is provided in fixed plate for the first groove of immobilized primer plate and the second groove for placing each centrifuge tube, the number of the second groove and the number of centrifuge tube corresponds；The third groove for placing multiple test tubes that plurality of target primer is housed is provided on primer plate, the number of third groove and the number of test tube correspond, and a test tube is equipped with a kind of target primer, and the lid of helixseal is equipped at test tube mouth.It include the primer plate for holding multiple test tubes of plurality of target primer in the kit, actually detected how many kinds of primer, how many a third grooves are just set on primer plate, place how many a test tubes, the diagnosis to multi-infection source can be achieved, ensure clinical precisely diagnosis pathogen infection type and adjustment pesticide application strategy, reduces mortality.

Description

A kind of kit for cerebrospinal fluid difficulty pathogen diagnosis

Technical field

This application involves medical field more particularly to a kind of kits for cerebrospinal fluid difficulty pathogen diagnosis.

Background technique

Currently, nervous centralis infection, encephalitis, meningitis most of are all as caused by various pathogen, therefore, to various The quick detection of pathogen is particularly important.Not so, higher case fatality rate, hospitalization cost rising and bed turnover day will be caused The health economics problems such as number increase.

Now, the main detection realized by diagnostic kit to the cerebrospinal fluid source of infection, still, traditional detection mode is only Can judge whether patient infects a kind of special pathogen, and can not determine patient whether the cause of disease also infected with other types Body, detectable infection source category is few, and diagnostic accuracy is lower, and then prevents diagnosis person from clinically accurate medication, can be wrong The golden hour of patient is spent, What is more will lead to patient and fortuitous event occur.

It can be seen that how increasing the detection type of the source of infection, improving the detection accuracy of the source of infection so that clinical accurate The problem of diagnosing pathogen infection type and adjustment pesticide application strategy, reducing mortality is that those skilled in the art are urgently to be resolved The problem of.

Utility model content

This application provides a kind of kit for cerebrospinal fluid difficulty pathogen diagnosis, solve in the prior art how Increase the detection type of the source of infection, improve the detection accuracy of the source of infection so that clinical precisely diagnosis pathogen infection type and tune The problem of whole pesticide application strategy, reduction mortality.

In order to solve the above technical problems, this application provides a kind of kit for cerebrospinal fluid difficulty pathogen diagnosis, Including reagent box body, further includes:

Positioned at the fixed plate of the reagent box body, primer plate and 96 hole PCR reaction plates；

The first groove for fixing the primer plate and for placing each centrifuge tube are provided in the fixed plate Two grooves, the number of second groove and the number of the centrifuge tube correspond；

It is provided with the third groove for placing multiple test tubes that plurality of target primer is housed on the primer plate, described the The number of three grooves and the number of the test tube correspond, and a test tube is equipped with a kind of target primer, the examination Pipe exit is equipped with the lid of helixseal.

Preferably, the sealing ring for sealing is additionally provided on the lid of the helixseal.

Preferably, each second groove is located at the same side of the fixed plate.

Preferably, each second groove is uniformly distributed in the same side of the fixed plate.

Preferably, the cross section of second groove and the third groove is circle.

Preferably, the cross section of the primer plate is rectangle；

Accordingly, the cross section of first groove is rectangle.

Preferably, further includes:

It is installed on the primer plate, the lid being engaged positioned at the edge of third groove side and the primer plate.

Preferably, the fixed plate and 96 hole reaction plate are laid in the bottom position of the reagent box body.

It preferably, further include etch layer on each centrifuge box and each test tube.

Preferably, the reagent box body is specially square.

Compared with the prior art, a kind of kit for cerebrospinal fluid difficulty pathogen diagnosis provided herein, is removed Include except reagent box body, further includes fixed plate, primer plate and the 96 hole PCR reaction plates positioned at reagent box body；Gu It is provided on fixed board for the first groove of immobilized primer plate and the second groove for placing each centrifuge tube, of the second groove Several numbers with centrifuge tube correspond；The for placing multiple test tubes that plurality of target primer is housed is provided on primer plate Three grooves, the number of third groove and the number of test tube correspond, and a test tube is equipped with a kind of target primer, test tube exit The lid of helixseal is installed.That is, including multiple test tubes for holding plurality of target primer in the kit Primer plate, actual needs detection how many kinds of primer (source of infection) is just arranged how many a third grooves on primer plate, and placement is more Few test tube, may be implemented the checkout and diagnosis to multi-infection source, improves the detection accuracy of the source of infection, and then may insure to face Bed precisely diagnoses pathogen infection type and adjustment pesticide application strategy, reduces mortality.

Detailed description of the invention

For the clearer technical solution for illustrating the application, letter will be made to attached drawing needed in the embodiment below The introduction wanted, it should be apparent that, it for those of ordinary skills, can be under the premise of not paying creativeness It obtains other drawings based on these drawings.

Fig. 1 is a kind of reagent cartridge configuration for cerebrospinal fluid difficulty pathogen diagnosis provided by the utility model embodiment Figure.

Specific embodiment

In order to make those skilled in the art better understand the technical solutions in the application, below in conjunction with attached drawing, it is right Technical solution in the embodiment of the present application carries out clear and complete description.

The core of the application is to provide a kind of kit for cerebrospinal fluid difficulty pathogen diagnosis, can solve existing skill How to increase the detection type of the source of infection in art, improve the detection accuracy of the source of infection so that clinical precisely diagnosis pathogen infection Type and adjustment pesticide application strategy, reduce mortality.

Fig. 1 is a kind of reagent cartridge configuration for cerebrospinal fluid difficulty pathogen diagnosis provided by the utility model embodiment Figure, as shown in Figure 1, the diagnostic kit includes reagent box body 1, further includes:

Positioned at the fixed plate 10 of reagent box body 1, primer plate 11 and 96 hole PCR reaction plates 12；

It is provided in fixed plate 10 for the first groove 100 of immobilized primer plate 11 and for placing each centrifuge tube 13 Two grooves 101, the number of the second groove 101 and the number of centrifuge tube 13 correspond；

The third groove 110 for placing multiple test tubes 14 that plurality of target primer is housed is provided on primer plate 11, the The number of three grooves 110 and the number of test tube 14 correspond, and a test tube 14 is equipped with a kind of target primer, 14 exit of test tube The lid 140 of helixseal is installed.

Specifically, reagent box body 1 mainly just refers to the shell of kit, can be carton box, is also possible to plastic casing. In the embodiment of the present application, the fixed plate 10 inside kit can select cystosepiment, be provided in fixed plate 10 for placing First groove 100 of primer plate 11, can make primer plate 11, completely or partially fixation is embedded in first to the size of the first groove 100 Subject to groove 100.The second groove 101 for placing each centrifuge tube 13 (EP pipe), centrifuge tube 13 are additionally provided in fixed plate 10 It is mainly used for holding Cerebrospinal Fluid in Patients, the reaction solution that the DNA extracting solution that extracts from the cerebrospinal fluid and when experiment need；In reality In the application of border, related personnel obscures the substance that each centrifuge tube 13 is filled in order to prevent, and then influences diagnostic result, it is preferable that each It further include etch layer on centrifuge tube 13, so as to the printed words of the engraving " this centrifuge tube is equipped with XX substance " in the etch layer, to improve Diagnostic accuracy；The number of second groove 101 and the number of centrifuge tube 13 correspond, that is, have several centrifuge tubes 13, just Several second grooves 101 are set in fixed plate 10, and the number of centrifuge tube 13 can be determined according to the actual situation, the second groove 101 size, which is subject to, can make all or part of fixation of each centrifuge tube 13 be embedded in the second groove 101；In view of the convenience of design The aesthetics of property and finished product, preferably embodiment, each second groove 101 are located at the same side of fixed plate 10, simultaneously Ensure that each second groove 101 is uniformly distributed in the same side of fixed plate 10, i.e., the same left or right side for being uniformly located at fixed plate 10, When the first groove 100 is located at the left side of fixed plate 10, each second groove 101 is uniformly located at the right side of fixed plate 10, when first When groove 100 is located at the right side of fixed plate 10, each second groove 101 is uniformly located at the left side of fixed plate 10, and certainly, each second is recessed Slot 101 can also be distributed in other ways in fixed plate 10, it is not limited to the distribution mode in the embodiment of the present application.In reality In the application of border, the size of the kit in the embodiment of the present application can be determined according to the actual situation, and the utility model is not made It limits.

Primer plate 11 inside kit can select rubber slab, be provided on primer plate 11 for placing multiple test tubes 14 Third groove 110, the number of test tube 14 needs the type of target primer according to actual needs to determine, a test tube 14 is equipped with one Kind of target primer, for example, if actual needs 29 kinds of primers of diagnosis, it is necessary to 29 test tubes 14, primer be also referred to as pathogen or The source of infection, the number of third groove 110 and the number of test tube 14 correspond, that is to say, that have several test tubes 14, it is necessary to Several third grooves 110 are set on primer plate 11, and the size of third groove 110 is can make all or part of fixed edge of each test tube 14 Subject to third groove 110, in actual design, third groove 110 can be made to be uniformly distributed in primer plate 11；Actually answering In, the primer filled in each test tube 14 is accurately quickly identified for the ease of related personnel, improves diagnosis efficiency, it is preferable that each It further include etch layer on test tube 14, so as to the printed words of the engraving " primer filled in this test tube 14 is XX " in the etch layer, to mention High diagnosis efficiency；In view of the characteristic of each target primer, 14 exit of test tube need to install the lid 140 of helixseal, and spiral is close The material of the lid 140 of envelope can be determined according to the actual situation.In view of the face shaping of centrifuge tube 13 and test tube 14, as It is preferably carried out mode, the cross section of the second groove 101 and third groove 110 is circle, certainly, and in practical applications, The cross section of two grooves 101 and third groove 110 can also be other shapes, as long as may insure that centrifuge tube 13 is all or part of It is capable of fixing the second groove 101 of insertion and all or part of be capable of fixing of test tube 14 is embedded in third groove 110.In view of setting The convenience of meter, preferably embodiment is in the cross section of primer plate 11 is rectangle；Accordingly, first groove 100 Cross section is rectangle, is which kind of shape particularly as the cross section for being primer plate 11, the cross section of the first groove 100 is just designed as Which kind of shape, i.e. saving material, are also convenient for immobilized primer plate 11.In view of the convenience of practical operation, preferably implement Mode, the hole of fixed plate 10 and 96 PCR reaction plate 12 is laid in the bottom position of reagent box body 1, it is, of course, also possible to will fix The hole of plate 10 and 96 PCR reaction plate 12, which stacks, to be placed, and the utility model is simultaneously not construed as limiting.In view of the rapidity and appearance of design Aesthetics, preferably embodiment, reagent box body 1 is specially square.Certainly, the specific shape of reagent box body 1 Shape is not limited to the shape in the embodiment of the present application, can also be designed to other shapes, and the utility model is simultaneously not construed as limiting.

It is typically provided with 96 reactive tanks 120 on 96 hole PCR reaction plates 12, is used to store reaction solution in reactive tank 120 (mixture of DNA extracting solution and plurality of target primer), the structure of 96 hole PCR reaction plates 12 and 96 hole PCR in the prior art The structure of reaction plate is the same, and for details, reference can be made to the prior art, details are not described herein for the utility model.

The condition of storage that the application implements provided kit is to be kept in dark place, and is avoided pollution, -20 DEG C of transports；-20 DEG C long-term preservation；Multigelation does not influence using effect；If frequently using, it is proposed that be stored in 4 DEG C, 4 DEG C can be reserved for 6 months.If Part solution generates precipitating, the solution in kit should be placed at room temperature for a period of time before use, when necessary can be in 37 DEG C of water 10min is preheated in bath, to dissolve precipitating.Points for attention are to need to read over this kit specification before experiment, in strict accordance with saying Bright book executes operation.It should illustrate that anhydrous second is added in elder generation in buffer and rinsing liquid according to reagent bottle label before first time use Alcohol.Multigelation should be avoided in sample, and the DNA fragmentation that otherwise will lead to extraction is smaller and extracted amount also declines.If buffer is clear There is precipitating in washing lotion, can re-dissolve in 37 DEG C of water-baths, be used after shaking up.All centrifugation steps are to use desk-top centrifugation Machine is centrifuged at room temperature.Sample should be balanced before bringing into operation to room temperature (15~25 DEG C).It is best to long-term preservation DNA sample The dehydrated alcohol of corresponding 2.5 times of volumes is added to precipitate DNA.White Flocculus can be seen after mixing.It is placed after of short duration centrifugation At least a year is saved in -80 DEG C of low temperature refrigerators.If needing using need to be centrifuged 5min in the centrifuge of 7000~8000 × g, make DNA is precipitated again, adds TE solution dissolving DNA precipitating.The purity and concentration of DNA must be detected again when necessary.MasterMix Should not multigelation, if be commonly used, preferably dissolve after be placed on 4 DEG C.After frozen product dissolution, it please turn upside down gently uniformly Mixing, is sure not excessively to exert oneself, avoids blistering, and use after gentle centrifugation.Reagent after melting please is placed on ice.Reaction is matched Set, dispense (free of contamination) pipette tips (be not loaded with the same pipette tips continuously), the miniature tube for having to renew etc., it avoids as far as possible Pollution.Gloves will be worn during using the kit, if gloves are contacted with sample, need to replace gloves immediately.

A kind of kit for cerebrospinal fluid difficulty pathogen diagnosis provided herein, in addition to including reagent box body Except, it further include fixed plate, primer plate and the 96 hole PCR reaction plates positioned at reagent box body；It is provided with and is used in fixed plate First groove of immobilized primer plate and the second groove for placing each centrifuge tube, the number of the second groove and the number of centrifuge tube It corresponds；The third groove for placing multiple test tubes that plurality of target primer is housed, third groove are provided on primer plate Number and the number of test tube correspond, test tube is equipped with a kind of target primer, and test tube exit is equipped with helixseal Lid.That is, include the primer plate for holding multiple test tubes of plurality of target primer in the kit, it is practical to need How many kinds of primer (source of infection) is detected, how many a third grooves are just set on primer plate, places how many a test tubes, Ke Yishi Now to the checkout and diagnosis in multi-infection source, the detection accuracy of the source of infection is improved, and then may insure clinical precisely diagnosis infection Pathogen type and adjustment pesticide application strategy, reduce mortality.

On the basis of Fig. 1, the kit further include: be installed on primer plate 11, be located at 110 side of third groove and primer The lid that the edge of plate 11 is engaged.Particularly as being to be additionally provided with lid on primer plate 11, lid can be with the edge of primer plate 11 Be interlocked, in practical applications, can by lid it is any on one side be fixedly installed in primer plate 11 while position place (because drawing The cross section of object plate 11 is rectangle, so the cross section of matched lid also should be rectangle), remaining other three While closely being fastened when lid is detained with the edge of primer plate 11, it can prevent test tube 14 in primer plate 11 and air long Time contact, can also shake in the third groove 110 on primer plate 11 to avoid test tube 14, if lid is light screening material, It can also play the role of being protected from light.In order to further prevent the air omitted to enter in test tube 14, the accuracy of diagnosis, In are influenced On the basis of above-described embodiment, preferably embodiment is additionally provided with for the close of sealing on the lid 140 of helixseal Seal.Particularly as being to add one layer of sealing ring again on the lid 140 of helixseal.

In order to make those skilled in the art more fully understand this programme, below in cerebrospinal fluid DNA extraction process and The diagnosis process of pathogen is described in detail.

First, paramagnetic particle method extracts the DNA in Cerebrospinal Fluid in Patients, specifically includes the following steps:

The first step collects 1mL determinand (cerebrospinal fluid) and 1.5mL centrifuge tube 13 is added.Second step is centrifuged at 9000rpm 30 seconds, cell precipitation is made to get off, abandons supernatant, be vortexed or flick and break up cell precipitation.Vortex oscillation until cell mass is sufficiently resuspended, Dispersion.The resuspension dispersion of cell is extremely important to cracking in next step, and cell, which is not broken up, is just added lysate, and will lead to cell cannot Sufficiently cracking forms naked eyes visible lumps.Third step, is added the cell that 350 μ l Lysis BufferA are resuspended, and piping and druming up and down is split Cell is solved, or the 10 seconds help lytic cells that are acutely vortexed, 70 DEG C of water-bath 10min.4th step (in practical operation, execute or Person does not execute the step not to be influenced on result is extracted), RNase A (10mg/ml) is added in lysate to 10 μ of final concentration G/ml, overturns 25 mixings, and 37 DEG C of incubation removals in 15 minutes remain RNA, then cool back room temperature.5th step is added ice-cold After 150 μ l SolutionAB, high speed continuous oscillation is mixed 25 seconds on turbula shaker, may be seen after mixing some small Protein mass.6th step, 12000rpm (can adjust as needed and increase centrifugal force) centrifugation 5 minutes, can see tube bottom at this time The lumpy precipitate of crineous or white, it is also possible to see some albumen precipitations and swim in liquid surface.7th step, it is careful to draw Supernatant (about 400 μ l) is into a new 1.5ml centrifuge tube 13.When drawing supernatant, be careful not to be drawn onto tube bottom and floating In the albumen precipitation of liquid surface.If be accidentally transferred to albumen precipitation in new centrifuge tube 13, after 2 minutes being centrifuged again Take supernatant.Isometric 400 μ l of room temperature BindingBuffer PQ and 20 μ l MagDNA magnetic beads is added in 8th step, at once on It overturns 20 times down and mixes well, at this time it is possible that flocculent deposit, it is also possible to will form the silk of one or similar double helix Shape substance cannot siphon away black clumps in practical operation.Centrifuge tube 13 is put into static 1- on magnetic separation rack by the 9th step 2min is clarified until solution becomes, and careful sops up liquid, not be drawn onto magnetic bead.Tenth step removes 1.5ml centrifuge tube 13, is added 500 μ l impurity removal buffer Wash Buffer B (please first check whether and dehydrated alcohol has been added) is mixed by inversion, by 1.5ml Centrifuge tube 13 be put into it is 1-2 minutes static on magnetic separation rack, until solution become clarify, carefully sop up liquid.11st step, adds Enter 600 μ l rinsing liquid Wash Buffer C (please first check whether and dehydrated alcohol has been added), be mixed by inversion, by 1.5ml centrifuge tube 13 are put into static 1-2min on magnetic separation rack, clarify until solution becomes, and careful sops up liquid, discards waste liquid.12nd step, 500 μ lWash Buffer D rinsing liquids (please first check whether and anhydrous second ferment has been added) are added, are mixed by inversion, by centrifuge tube 13 It is put into static 1-2min on magnetic separation rack, is clarified until solution becomes, careful sops up liquid, discards waste liquid.It (inhales as far as possible Fall residual liquid, in order to avoid influence experimental result).13rd step removes 1.5ml centrifuge tube 13, places it in 37-65 DEG C of baking Residual ethanol is dried in case.(2-3min can also be blown with hair dryer, should not be too dry, in order to avoid DNA is difficult to dissolve).14th Step is added 50-100 μ l elution buffer gently springing tube wall, magnetic bead is broken up completely and is dispersed in elution buffer, room temperature is put Setting 1-2min, (elution buffer pre-heat effect in 65-70 DEG C of water-bath is more preferable, if the DNA that obtain higher concentration can subtract Few elution volume, can also elute in two times).Centrifuge tube 13 is put into static 1-2mim on magnetic separation rack by the 15th step, until Solution becomes clarification, and careful transfer solution not be drawn onto magnetic bead, if being drawn onto magnetic bead can relay into a new centrifuge tube 13 It sets and is separated on magnetic frame.16th step after obtaining DNA extracting solution, can be deposited under 2-8 DEG C of environment, if wanted It stores, can be placed under -20 DEG C of environment for a long time.

Second, the DNA purity of extraction is identified.

Because the OD260/OD280 of pure DNA/RNA is 1.8 or 2.0, therefore can be estimated according to the ratio of OD260/OD280 Count out the purity of DNA.If the higher explanation of ratio contains RNA, the lower explanation of ratio is with the presence of residual protein.OD230/OD260 Ratio should be between 0.4-0.5, if the higher explanation of ratio is with the presence of remaining salt.Specifically, if the ratio of OD260/OD280 R is greater than 1.8, illustrates there are RNA, can be handled again with RNaseA.If R value less than 1.8, illustrates have the impurity such as protein to deposit , need to again with Proteinase K, SDS and expect, chloroform, isoamyl alcohol again purify to DNA (3M of 1/8 volume can also be added NaAc (pH5.2) promotes DNA Precipitation together with cold ethyl alcohol.

Second, the source of infection in Cerebrospinal Fluid in Patients is detected.

The first step configures primer solution.(4000rpm, 30~60s) is please first centrifuged before uncapping；Slowly open on test tube 14 Helixseal lid 140, suitable buffer solution or water is added；Screw on fully shake after the lid 140 of helixseal it is mixed It is even；Do not have to temporarily or long term storage needs are placed on -20 DEG C of refrigerations.

Second step, firstly, configuring PCR reaction solution in the way of table 1.

1 PCR reaction solution of table proportion

Agent formulations	Usage amount	Usage amount	Usage amount
				qPCR MasterMix	10ul	12.5ul	25ul
PCR forward primer	0.5ul	0.5ul	1ul
				PCR reverse primer	0.5ul	0.5ul	1ul
DNA profiling	1ul	1ul	2ul
				DdH2O (sterile purified water)	8.0ul	10.5ul	21ul
It amounts to	20ul	25ul	50ul

In 20ul reaction system, the additive amount of DNA profiling is usually in 100ng hereinafter, because in different types of DNA profiling The copy number of the target gene contained is different, can carry out gradient dilution when necessary, determine optimal DNA profiling additive amount.If be intended to The second step pcr amplification reaction of 2 step Real Time-PCR reaction is carried out using this product, the reaction solution of the first step is as DNA mould Additive amount when plate does not exceed the 10% of PCR reaction solution total volume.96 orifice plates use 50ul reaction system.

Secondly, carrying out Real Time-PCR reaction.Solubilising reagent, mixing of turning upside down, confirmation reaction solution are completely dissolved (avoiding mixing using oscillator, acutely oscillation can reduce properties of product)；By configured PCR reaction mixture, packing to PCR In reaction tube；Add the template of PCR amplification；Pcr amplification reaction is carried out using Real Time PCR amplification instrument；Reaction solution is matched It sets method and PCR amplification condition please refers to application method；The application method of Real Time PCR instrument, please refers to corresponding instrument Specification.

In practical applications, the three-step approach PCR response procedures that following display can be used, which diagnose, determines the source of infection, if the journey Sequence cannot get good experimental result, can carry out the optimization of PCR condition again.Three-step approach PCR amplification standardization program is as follows:

1, initial denaturation: 95 DEG C of 2min.2, PCR reacts, and 10s is carried out under 95 DEG C of environment, 30- is carried out under 56 DEG C of environment 34s carries out 30s under 72 DEG C of environment, and the process is recyclable to be executed 40 times.Annealing temperature can be set according to primer.3, melt Curve carries out 1min under 95 DEG C of environment, and 1min is carried out under 55 DEG C of environment, and 86 circulations are carried out under 55 DEG C of -98 DEG C of environment.

Finally, Real Time-PCR data are analyzed.The amplification curve of Real Time-PCR is confirmed after reaction and is melted Solution curve carries out making standard curve etc. when PCR is quantitative.The specific effect using following assessment real-time quantitative PCR reaction, 1, PCR amplification efficiency at least needs to do 3 parallel repetitions, at least does 5 orders of magnitude to correctly assess PCR amplification efficiency Multiple (5logs) continuous gradient dilutions template concentrations.2, R2 value, it is to illustrate that the statistics of degree of correlation between two values is academic Language.If R2 is equal to 1, can be with Y value (Ct) come Accurate Prediction X value (amount).If R2 be equal to 0, cannot by Y value come Predict X value.When R2 value is greater than 0.99, relevant confidence level is fine between two values.3, accuracy, standard deviation be (deviation Square root) it is most common accuracy metering method.If many data points are all close to average value, standard deviation is with regard to small； If many data points are all far from average value, standard deviation is with regard to big.In fact, the data group meeting that enough numbers of repetition generate Form rough normal distribution.This can often prove that I.i.d. random variables exist by classical central limit theory It is intended to normal distribution when unlimited more.If PCR reaction efficiency is 100%, the average interval Ct between 2 times of dilution points is answered This is just 1 Ct value.2 times of diluted concentrations are differentiated with 99.7% probability, standard deviation is just necessarily less than equal to 0.167.Mark Quasi- deviation is bigger, and it is lower to differentiate 2 times of diluted abilities.In order to tell 2 times of dilutions in the case where 95% or more, mark Quasi- deviation is necessarily less than equal to 0.250.4, sensitivity, no matter CT absolute value is how many, any effectively to expand and detect The system that beginning template copy numbers are 1 has all reached the limit of sensitivity.PCR efficiency is to determine the key factor of reaction sensitivity. Another important consideration factor when detecting extremely low copy number is, template number when low-copy cannot be come by general case It is expected that.On the contrary, it can follow Poisson distribution, that is, carry out a large amount of parallel starting mould that repeats, should averagely copy containing one Plate, actually about 37% without containing copy, and only about 37% containing 1 copy, and about 18% actually containing there are two copies.Cause This, in order to more reliably detect low-copy, it is necessary to do a large amount of parallel repetition experiment to provide statistical significance, and overcome Poisson The limitation of distribution.Evaluate the standard of fluorescent PCR result.

Particularly as being to instill DNA extracting solution and corresponding target in the reactive tank 120 on 96 hole PCR reaction plates 12 to draw Object instills a kind of target primer in one reactive tank 120, then, passes through relevant detecting instrument or mesh that method detection instills Whether index object is positive in the reaction solution that current reactive tank 120 is held, if it is, illustrating that corresponding patient infects The primer illustrates to correspond to if whether the target primer instilled is negative in the reaction solution that current reactive tank 120 is held Patient do not infect the primer, repeat this step, completed until all target primer is detected, when determining to work as After the source of infection of preceding patient, so that it may accurately carry out medication to the patient, improve the therapeutic efficiency of patient.In the embodiment of the present application In have chosen 25 kinds of primers (source of infection or bacterium), be specifically shown in Table 2, the details of 25 kinds of primers is as shown in table 2.Wherein, it wraps Include 14 kinds of bacteriums, 7 kinds of fungies, 4 kinds of viruses.

The details of each bacterium of table 2

Those skilled in the art will readily occur to its of the application after considering specification and practicing application disclosed herein His embodiment.This application is intended to cover any variations, uses, or adaptations of the application, these modifications, purposes or Person's adaptive change follow the general principle of the application and include common knowledge in the art disclosed in the present application or Conventional techniques.Description and embodiments are considered only as exemplary, and the just true range of the application is pointed out by claim.

It should be understood that the application is not limited to the precise structure that has been described above and shown in the drawings, and And it can carry out various modifications and change without departing from the scope again.Above-described the application embodiment is not constituted to this Shen Please protection scope restriction.

Claims (10)

1. a kind of kit for cerebrospinal fluid difficulty pathogen diagnosis, including reagent box body, which is characterized in that further include:

Positioned at the fixed plate of the reagent box body, primer plate and 96 hole PCR reaction plates；

The first groove for fixing the primer plate and second for placing each centrifuge tube recessed is provided in the fixed plate Slot, the number of second groove and the number of the centrifuge tube correspond；

The third groove for placing multiple test tubes that plurality of target primer is housed is provided on the primer plate, the third is recessed The number of slot and the number of the test tube correspond, and a test tube is equipped with a kind of target primer, and the test tube goes out The lid of helixseal is installed at mouthful.

2. the kit according to claim 1 for cerebrospinal fluid difficulty pathogen diagnosis, which is characterized in that the spiral The sealing ring for sealing is additionally provided on the lid of sealing.

3. the kit according to claim 1 for cerebrospinal fluid difficulty pathogen diagnosis, which is characterized in that each described Two grooves are located at the same side of the fixed plate.

4. the kit according to claim 3 for cerebrospinal fluid difficulty pathogen diagnosis, which is characterized in that each described Two grooves are uniformly distributed in the same side of the fixed plate.

5. the kit according to claim 1 for cerebrospinal fluid difficulty pathogen diagnosis, which is characterized in that described second The cross section of groove and the third groove is circle.

6. the kit according to claim 1 for cerebrospinal fluid difficulty pathogen diagnosis, which is characterized in that the primer The cross section of plate is rectangle；

Accordingly, the cross section of first groove is rectangle.

7. the kit according to claim 6 for cerebrospinal fluid difficulty pathogen diagnosis, which is characterized in that further include:

It is installed on the primer plate, the lid being engaged positioned at the edge of third groove side and the primer plate.

8. the kit according to claim 1 for cerebrospinal fluid difficulty pathogen diagnosis, which is characterized in that the fixation Plate and 96 hole reaction plate are laid in the bottom position of the reagent box body.

9. the kit according to claim 1 for cerebrospinal fluid difficulty pathogen diagnosis, which is characterized in that it is each it is described from It further include etch layer on heart pipe and each test tube.

10. according to claim 1 to the kit for being used for cerebrospinal fluid difficulty pathogen diagnosis described in 9 any one, feature It is, the reagent box body is specially square.