CN103255216A - Nosocomial infection multiple causative agent parallel detection gene chip and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical fields of molecular biological techniques and clinical tests, and particularly discloses a nosocomial infection multiple causative agent parallel detection gene chip and a preparation method and application thereof. The gene chip can be used for rapidly and sensitively detecting fourteen medical treatment infection typical causative agents such as acinetobacter baumannii, enterobacter cloacae, enterococcus faecalis and the like by using one reaction, and the detection data of coinfection of yeast and bacterium which cannot be provided by a traditional amplification method is offered. Furthermore, the gene chip also comprises a methicillin resistance target spot, thus realizing further detection of staphy lococcus infection, therefore, a clinician can realize which causative agent a patient is infected in one or two hours, and realize whether the patient has drug resistance, so as to decide whether the patient needs antibiotics or which antibiotics is needed, so that the gene chip has a greater clinical guiding significance.

Description

Many pathogenic agent of ward infection parallel detection gene chip and preparation method thereof and application

(1) technical field

The invention belongs to Protocols in Molecular Biology and clinical detection technique field, particularly a kind of many pathogenic agent of ward infection parallel detection gene chip and preparation method thereof and application.

(2) background technology

In recent years, along with the development of medical science, incidence of nosocomial infection is in rising trend, has caused the attention of countries in the world hospital, and therefore, ward infection is proposed surveillance and control measure has become a comprehensive study problem.External advanced country China's morning carried out the research of ward infection in 30 years, China's incidence of nosocomial infection is 9%～20%, China administrative departments of public health at different levels very pay attention to, the Ministry of Health has only set up 134 hospitals in the whole nation with 3 years and has participated in the ward infection monitor network, and the ward infection monitoring standard is listed in the enforcement of hospital's split pole management, utilize the means of management monitoring to promote standards such as ward infection characteristic analyzing and testing.

For ward infection, how the existing diagnostic techniques of hospital is by bacterium or virus culture, and cultivating needs the time, and normally one day or several days, so general doctor does not wait cultivation results to come out with regard to the medication of empirical ground.The result who does like this has the medication more than 50% inaccurate, incurs loss through delay real treatment time, and patient is unnecessarily prolonged the hospital stays, has increased medical treatment cost.The diagnosis of this " wise afterwards " formula also is compelled to do, and does not singly do cultivation because if the doctor does not open, and the malpractice that sing misdiagnosis and mistreatment produces is just responsible by hospital and doctor.Have no alternative when disconnected experiment is single so the doctor of Infectious Disease begins to treat patients, opened, this patient there is not real help, only be in order to record one in the medical archive the inside, in case defendant and medical tangle from now on, this is called " Defensive medicine " or " defence medical science ", be not active attack disease, but the prevention patient accuses the doctor.Such diagnosis not only can not be lowered medical expense, allows expense constantly increase on the contrary.The diagnostic techniques that falls behind is the reason of medical expense sustainable growth, and new faster more accurate molecule differential diagnosis technology then can allow hospital reduce cost, and also reduces medical treatment to entire society and consumes, and reduces legal dispute.

The iCubate technology platform integrates nucleic acid extraction, amplification and three steps of detection, and multiple, automatic, sealing (anti-pollution) these three clinical key functions that press for are provided, and avoids polluting and the false positive that causes.Use this technology platform, the molecular diagnosis of a plurality of target spots can be finished in one and a half hours at one hour.

The detection of ward infection is put on the iCubate technology platform, can accomplishes a sample, a reaction, a plurality of indexs.Whether, be other pathogenic infection, whether resistance is arranged if so just can know whether patient has concurrent infection, be applied to clinically, can instruct the clinician to determine whether this usefulness or use which kind of antibiotic of patient, just bigger to clinical directive significance.

(3) summary of the invention

The present invention is in order to remedy the deficiencies in the prior art, provides a kind of and detected rapid sensitive, many pathogenic agent of ward infection parallel detection gene chip that accuracy is high and preparation method thereof and use.

The present invention is achieved through the following technical solutions:

A kind of many pathogenic agent of ward infection parallel detection gene chip, main by the gene chip that related diseases substance specific probe is fixedly arranged, Auele Specific Primer and universal primer are formed, it is characterized in that: described related diseases substance comprises Bao formula acinetobacter calcoaceticus, streptococcus aureus, Pseudomonas aeruginosa, pneumobacillus, enterococcus faecalis, faecium, intestinal bacteria, enterobacter cloacae, Proteus mirabilis, Candida albicans, candida krusei, Candida glabrata, Candida parapsilosis and Oidium tropicale be totally 14 kinds of pathogenic agent, also comprises an X-1497 resistance target spot in addition; Described gene chip is dot matrix and above-mentioned 15 kinds of pathogen specific probes is fixedly arranged that described Auele Specific Primer can carry out specific amplification to above-mentioned 15 kinds of pathogenic agent target genes; Described universal primer is the irrelevant nucleotide sequences of above-mentioned 15 kinds of pathogenic agent target genes, and wherein oppositely universal primer 5 ' end has fluorescent mark.

The present invention can primary first-order equation simultaneously to Acinetobacter baumannii, enterobacter cloacae, 14 kinds of medical treatment such as enterococcus faecalis are infected typical pathogenic agent and are carried out fast, sensitive detection, yeast that traditional amplification method can't provide and the detection data of bacterium coinfection are provided, also comprise an X-1497 resistance target spot in addition, thereby can further realize the detection to staphylococcal infections, can allow the clinician just can know that in one or two hour which kind of pathogenic infection patient is like this, whether resistance is arranged, thereby determine which kind of antibiotic whether patient should use or use, just bigger to clinical directive significance.

It is as follows to detect the pathogenic agent index:

15 kinds of probes in the described gene chip are:

Acinetobacter baumannii: 5 '-AAG TGT AGC TCA TCT TGA AG-3 ';

Streptococcus aureus: 5 '-ACT TAA TGA CTC TCA AGC T-3 ';

Pseudomonas aeruginosa: 5 '-ACT CCA CCG A CT CGA TCG TC-3 ';

Pneumobacillus: 5 '-ACG TGC TGC CCG CCT ATG AA-3 ';

Enterococcus faecalis: 5 '-TTA ATA TTT TAA ACA AAG AT-3 ';

Faecium: 5 '-ATG ATA TTG AGT TAG ATG AA-3 ';

Intestinal bacteria: 5 '-AGG TTA AAG CGA CTG AGG TC-3 ';

Enterobacter cloacae: 5 '-GGA TAT CAA A CC GTT CTT TA---3 ';

Proteus mirabilis: 5 '-ATT ATG ATG AGC CAA AAA AT-3 ';

Candida albicans: 5 '-GAA CAA AAA A AA TTT GCT AA-3 ';

Candida krusei: 5 '-AAG AAG ATT GGT TTT CTT GA-3 ';

Candida glabrata: 5 '-AAG AAA TCT TGT GTT GAC TG-3 ';

Candida parapsilosis: 5 '-GTG CCA TTG ACA ATG TAT GT-3 ';

Oidium tropicale: 5 '-CCA TAA TCA ACA GTA TCT TC-3 ';

X-1497 resistance: 5 '-ATT AAC AGC A AT GAT TGG GT---3 '.

The sequence of described Auele Specific Primer is as follows:

Acinetobacter baumannii: 5 '-GCG AGC TTC CAG AAC AAA GA,

5’—GAT?GCG?CTT?GCA?TAT?GTG?GT；

Streptococcus aureus: 5 '-CAC GAT GAA GCT CAA CAA AAT GC,

5’—TGT?TGC?GCA?TCA?GCT?TTT?GGA；

Pseudomonas aeruginosa: 5 '-GAG TGG CAA CGT ACA CCT CA,

5’—CCG?GCA?CGT?ATA?CCA?CAA?GA；

Pneumobacillus: 5 '-GCT GTT TAA CAA CCA GGC CG,

5’—CGG?GGT?ATA?GTT?GCG?CTC?TT；

Enterococcus faecalis: 5 '-ATG GGG CCA GAA AAG CTC G,

5’—GCC?GCA?TCA?TTG?TGT?TCC?C；

Faecium: 5 '-TGA CTC GGG GAT TGA TAG GC,

5’—ACT?CTT?GGA?GCA?ATC?GTG?TCT?G；

Intestinal bacteria: 5 '-AAG CGA CTG TTA GTG CGA CA,

5’—CAT?CAC?CAC?CGC?TTG?CTT?TC；

Enterobacter cloacae: 5 '-TGC TGA TCA CCG GGG TTT AC,

5’—CAG?CAG?GTC?GTC?CGT?CAT?AA；

Proteus mirabilis: 5 '-GCG GTT TAT CAC GAA GGG GT,

5’—ATT?GGC?AAA?ACG?AAA?CGC?CC；

Candida albicans: 5 '-CCA GTT TTC GGT ACA GGG GT,

5’—ACA?TTG?GCA?ACC?CCA?TGA?GT；

Candida krusei: 5 '-TGT CTT TGC CTA TGC CGG TT,

5’—CAG?ACC?ACC?CAA?AGG?CAT?GA；

Candida glabrata: 5 '-GCA TTT CGC TGC GTT CTT CA,

5’—GTT?GGG?GGA?ACG?CTC?TCT?TT；

Candida parapsilosis: 5 '-GTG TCG GAC CCA ATG GAT GT,

5’—ATA?CCC?ACC?ATC?GTA?CCC?CA；

Oidium tropicale: 5 '-CCA CCG AAT GGC AAG TAT GGA,

5’—ACC?AAG?GCT?AGT?GCT?GTT?TC；

The X-1497 resistance: 5 '-ACC TCT GCT CAA CAA GTT CCA,

5’—ACG?TTG?TAA?CCA?CCC?CAA?GA；

More than all Auele Specific Primers 5 ' end all have a universal primer label, the universal primer sequence is as follows:

5’—ACG?TGT?TAC?TCG?CAT?AGT?CG，

5’—TCC?TGT?ACT?CTC?TTA?CTG?AG。

The making method of many pathogenic agent of ward infection parallel detection gene chip of the present invention comprises the steps:

(1) probe design is according to the conservative fragments designing probe of 15 kinds of pathogen gene groups, length 20bp;

(2) preparation of chip, probe is synthetic, earlier add the poly-poly T of the preceding paragraph 8-20 at as above 5 ' end of 15 probes, carry out amido modifiedly simultaneously,, and mix with spotting solution equal-volume probe dilution with deionized water, make that final concentration is 75 pmol/ μ l, in aldehyde group modified slide surface, place 75% relative humidity by cartesian microarray manufacturing system dot matrix, fixed in 48-72 hour under the room temperature condition; After the taking-up slide is put in concussion washing 2 min among 0.2% SDS, takes out fully drying of back room temperature, then slide is put in concussion 5 min in the sodium borohydride solution, to remove free aldehyde radical; With 0.2% SDS concussion washing, 1 min, repeat twice, soak 2 min with pure water at last, dry; So, many pathogenic agent of ward infection parallel detection gene chip of 15 probes is namely made and is finished, and the hybridization that then chip is fixed on the cartridge bottom detects in the groove.

The application of many pathogenic agent of ward infection parallel detection gene chip of the present invention in hospital, clinic or other hygienic measures sanatorium ward infection clinical diagnosises mainly comprises the steps:

(1) chip preparation: with 15 kinds of ward infection pathogen specific probe dot matrix and be fixed on the aldehyde group modified slide glass, the chip preparation hybridization that is fixed in the cartridge bottom that finishes detects in the groove;

(2) separate nucleic acid: use business-like test kit to extract RNA;

(3) pcr amplification: use Auele Specific Primer and universal primer to carry out pcr amplification reaction;

(4) hybridization: the liquid that moves of cartridge inside was robbed and all products all are put into hybridization are detected groove and hybridize after pcr amplification was finished, during hybridization the well heater of chip bottom with temperature control at 65 ℃.Moving liquid after hybridization in 15 minutes finishes robs and does Rapid Cleaning;

(5) signal detection: the cartridge of having cleaned is put into the last signal detection of acceptance in the cartridge reading apparatus, there are proofing units such as laser confocal scanning system and photomultiplier cartridge reading apparatus the inside, the data of reading directly manifest with the form of numeral by the systems analysis software processes, cartridge is just taken out from reading apparatus and is directly thrown away, and closed system has been avoided the generation of polluting.

Product of the present invention can carry out somatotype detection accurately to 14 kinds of pathogenic agent that cause ward infection, and specificity and good reproducibility can be used as the examination of ward infection pathogenic agent, detect rapid sensitive, and accuracy is strong, is suitable for wide popularization and application.

(4) embodiment

Embodiment 1: the extraction of sample nucleic acid

Use QIGEN Viral RNA mini Extraction Kit (CAT:52904) from clinical samples, to extract RNA.Extract 43 parts of sample nucleic acid altogether, wherein 28 parts is the known positive sample of hypotype, 10 parts of negative contrasts, and 5 parts is uncorrelated Virus Sample.Sample information such as following table:

(1) adds the AVL damping fluid of 560 μ l in the 1.5ml centrifuge tube;

(2) in this centrifuge tube, add 140 μ l clinical samples again;

(3) place 10min under the room temperature, instantaneous centrifugal then;

(4) add 560 μ l dehydrated alcohols, abundant mixing is 15s at least, and is instantaneous centrifugal;

(5) mixing solutions that carefully adds about 630 μ l is to the QIAamp post, and on collection tube, the centrifugal 5s of 8000rpm makes liquid pass through filter membrane then with the QIAamp column sleeve;

(6) discard liquid in the collection tube, repeat the 6th step;

(7) discard the AW1 damping fluid of the careful 750 μ l of adding of liquid in the collection tube, the centrifugal 5s of 8000rpm makes liquid pass through filter membrane;

(8) discard the AW2 damping fluid of the careful 750 μ l of adding of liquid in the collection tube, the centrifugal 5s of 8000rpm makes liquid pass through filter membrane;

(9) discard liquid in the collection tube, 13000rpm recentrifuge 1min;

(10) the QIAamp post is put into the centrifuge tube of a clean 1.5ml;

(11) add the EB damping fluid of 30 μ l on the film, leave standstill 2-5min under the room temperature then;

(12) in whizzer with the centrifugal 1min of the speed of 8000rpm, the centrifugal liquid that gets off is required nucleic acid solution.

Embodiment 2:PCR amplification and hybridization

(cartridge detects a sample, also is provided with a blank as required in addition with above-mentioned sample nucleic acid.Repeatability for detection reagent, each sample is done three repetitions) add hand-hole from sample and join the cartridge, pcr amplification, hybridization detection are housed in the cartridge in advance, clean required all reagent, the hybridization that the ward infection pathogen gene chip for preparing also is placed on the cartridge bottom in advance detects in the groove.Cartridge is put in the cartridge processing instrument, from system controlling software, writes response procedures, operation, instrument carries out pcr amplification, hybridization automatically.

(1) pcr amplification carries out two-wheeled, and the reagent that first round pcr amplification adopts is Qiagen single stage method RT-PCR Kit, Cat No. 210212, and reaction system disposes as following table:

Response procedures is as follows:

Second reagent of taking turns the pcr amplification employing is Qiagen multiplex PCR Kit, Cat No. 206143, and reaction system disposes as following table:

Response procedures is as follows:

(2) hybridization:

The liquid that moves of cartridge inside was robbed and all products all are put into hybridization are detected groove and hybridize after pcr amplification was finished.The well heater of chip bottom controls temperature 65 during hybridization ℃Moving liquid after hybridization in 15 minutes finishes robs and does Rapid Cleaning.The cartridge of having cleaned is put into the last signal detection of acceptance in the cartridge reading apparatus.

More than all reagent, comprise: the corresponding reagent groove that nucleic acid extraction, pcr amplification, hybridization, cleaning etc. all are placed on bottom the cartridge in advance is interior (for reducing cost, also can use commercialization to extract test kit manual extraction nucleic acid, nucleic acid joined in the cartridge gets final product), realize that by the pipettor in the cartridge accurate liquid shifts during application.Cartridge is totally-enclosed design, has avoided crossed contamination and high density product pollution.

Amplification, hybridization program write in system controlling software in advance, and the cartridge processing instrument is used " conversion thermostatically heating head " amplification technique, realizes rapid amplifying, hybridization automatically by 3 fixed temperature heating heads of changing fast.

Embodiment 3: signal detection

Reaction finishes, cartridge is taken out from processing instrument, be put in the cartridge reading apparatus, there are proofing units such as laser confocal scanning system and photomultiplier cartridge reading apparatus the inside, the data of reading directly display with the form of numeral by the systems analysis software processes, cartridge is just taken out from reading apparatus and is directly thrown away, and closed system has been avoided the generation of polluting.Detected result is as follows:

Interpretation of result:

(1) this reagent can carry out accurate somatotype to 28 parts of positive bacteria sample of nucleic acid, and specificity is up to 100%.

(2) good reproducibility, 90% detection batch within variance coefficient (CV)＜10%.

(3) detect 10 parts of known negative sample, all there is not false positive in result's demonstration.

(4) detect 5 parts of other Virus Samples, the result shows all negative, does not have the situation of false retrieval.

The detected result explanation: this reagent can carry out somatotype detection accurately to 14 kinds of pathogenic agent that cause ward infection, and specificity and repeatability are all good.Further after the clinical verification, can be used as the examination of the susceptible substance of institute.

Claims (6)

1. many pathogenic agent of ward infection parallel detection gene chip, main by the gene chip that related diseases substance specific probe is fixedly arranged, Auele Specific Primer and universal primer are formed, it is characterized in that: described related diseases substance comprises Bao formula acinetobacter calcoaceticus, streptococcus aureus, Pseudomonas aeruginosa, pneumobacillus, enterococcus faecalis, faecium, intestinal bacteria, enterobacter cloacae, Proteus mirabilis, Candida albicans, candida krusei, Candida glabrata, Candida parapsilosis and Oidium tropicale be totally 14 kinds of pathogenic agent, also comprises an X-1497 resistance target spot in addition; Described gene chip is dot matrix and above-mentioned 15 kinds of pathogen specific probes is fixedly arranged that described Auele Specific Primer can carry out specific amplification to above-mentioned 15 kinds of pathogenic agent target genes; Described universal primer is the irrelevant nucleotide sequences of above-mentioned 15 kinds of pathogenic agent target genes, and wherein oppositely universal primer 5 ' end has fluorescent mark.

2. many pathogenic agent of ward infection parallel detection gene chip according to claim 1 is characterized by, and 15 kinds of probes in the described gene chip are:

Acinetobacter baumannii: 5 '-AAG TGT AGC TCA TCT TGA AG-3 ';

Streptococcus aureus: 5 '-ACT TAA TGA CTC TCA AGC T-3 ';

Pseudomonas aeruginosa: 5 '-ACT CCA CCG A CT CGA TCG TC-3 ';

Pneumobacillus: 5 '-ACG TGC TGC CCG CCT ATG AA-3 ';

Enterococcus faecalis: 5 '-TTA ATA TTT TAA ACA AAG AT-3 ';

Faecium: 5 '-ATG ATA TTG AGT TAG ATG AA-3 ';

Intestinal bacteria: 5 '-AGG TTA AAG CGA CTG AGG TC-3 ';

Enterobacter cloacae: 5 '-GGA TAT CAA A CC GTT CTT TA---3 ';

Proteus mirabilis: 5 '-ATT ATG ATG AGC CAA AAA AT-3 ';

Candida albicans: 5 '-GAA CAA AAA A AA TTT GCT AA-3 ';

Candida krusei: 5 '-AAG AAG ATT GGT TTT CTT GA-3 ';

Candida glabrata: 5 '-AAG AAA TCT TGT GTT GAC TG-3 ';

Candida parapsilosis: 5 '-GTG CCA TTG ACA ATG TAT GT-3 ';

Oidium tropicale: 5 '-CCA TAA TCA ACA GTA TCT TC-3 ';

X-1497 resistance: 5 '-ATT AAC AGC A AT GAT TGG GT---3 '.

3. many pathogenic agent of ward infection parallel detection gene chip according to claim 1 is characterized by, and the sequence of described Auele Specific Primer is as follows:

Acinetobacter baumannii: 5 '-GCG AGC TTC CAG AAC AAA GA,

5’—GAT?GCG?CTT?GCA?TAT?GTG?GT；

Streptococcus aureus: 5 '-CAC GAT GAA GCT CAA CAA AAT GC,

5’—TGT?TGC?GCA?TCA?GCT?TTT?GGA；

Pseudomonas aeruginosa: 5 '-GAG TGG CAA CGT ACA CCT CA,

5’—CCG?GCA?CGT?ATA?CCA?CAA?GA；

Pneumobacillus: 5 '-GCT GTT TAA CAA CCA GGC CG,

5’—CGG?GGT?ATA?GTT?GCG?CTC?TT；

Enterococcus faecalis: 5 '-ATG GGG CCA GAA AAG CTC G,

5’—GCC?GCA?TCA?TTG?TGT?TCC?C；

Faecium: 5 '-TGA CTC GGG GAT TGA TAG GC,

5’—ACT?CTT?GGA?GCA?ATC?GTG?TCT?G；

Intestinal bacteria: 5 '-AAG CGA CTG TTA GTG CGA CA,

5’—CAT?CAC?CAC?CGC?TTG?CTT?TC；

Enterobacter cloacae: 5 '-TGC TGA TCA CCG GGG TTT AC,

5’—CAG?CAG?GTC?GTC?CGT?CAT?AA；

Proteus mirabilis: 5 '-GCG GTT TAT CAC GAA GGG GT,

5’—ATT?GGC?AAA?ACG?AAA?CGC?CC；

Candida albicans: 5 '-CCA GTT TTC GGT ACA GGG GT,

5’—ACA?TTG?GCA?ACC?CCA?TGA?GT；

Candida krusei: 5 '-TGT CTT TGC CTA TGC CGG TT,

5’—CAG?ACC?ACC?CAA?AGG?CAT?GA；

Candida glabrata: 5 '-GCA TTT CGC TGC GTT CTT CA,

5’—GTT?GGG?GGA?ACG?CTC?TCT?TT；

Candida parapsilosis: 5 '-GTG TCG GAC CCA ATG GAT GT,

5’—ATA?CCC?ACC?ATC?GTA?CCC?CA；

Oidium tropicale: 5 '-CCA CCG AAT GGC AAG TAT GGA,

5’—ACC?AAG?GCT?AGT?GCT?GTT?TC；

The X-1497 resistance: 5 '-ACC TCT GCT CAA CAA GTT CCA,

5’—ACG?TTG?TAA?CCA?CCC?CAA?GA；

More than all Auele Specific Primers 5 ' end all have a universal primer label, the universal primer sequence is as follows:

5’—ACG?TGT?TAC?TCG?CAT?AGT?CG，

5’—TCC?TGT?ACT?CTC?TTA?CTG?AG。

4. the making method of many pathogenic agent of ward infection parallel detection gene chip according to claim 1 is characterized by, and comprises the steps:

(1) probe design is according to the conservative fragments designing probe of 15 kinds of pathogen gene groups, length 20bp;

(2) preparation of chip, probe is synthetic, earlier add the poly-poly T of the preceding paragraph 8-20 at as above 5 ' end of 15 probes, carry out amido modifiedly simultaneously,, and mix with spotting solution equal-volume probe dilution with deionized water, make that final concentration is 75 pmol/ μ l, in aldehyde group modified slide surface, place 75% relative humidity by cartesian microarray manufacturing system dot matrix, fixed in 48-72 hour under the room temperature condition; After the taking-up slide is put in concussion washing 2 min among 0.2% SDS, takes out fully drying of back room temperature, then slide is put in concussion 5 min in the sodium borohydride solution, to remove free aldehyde radical; With 0.2% SDS concussion washing, 1 min, repeat twice, soak 2 min with pure water at last, dry; So, many pathogenic agent of ward infection parallel detection gene chip of 15 probes is namely made and is finished, and the hybridization that then chip is fixed on the cartridge bottom detects in the groove.

5. the application of many pathogenic agent of ward infection parallel detection gene chip according to claim 1 in hospital, clinic or other hygienic measures sanatorium ward infection clinical diagnosises.

6. the application of gene chip according to claim 5 is characterized by, and mainly comprises the steps:

(1) chip preparation: with 15 kinds of ward infection pathogen specific probe dot matrix and be fixed on the aldehyde group modified slide glass, the chip preparation hybridization that is fixed in the cartridge bottom that finishes detects in the groove;

(2) separate nucleic acid: use business-like test kit to extract RNA;

(3) pcr amplification: use Auele Specific Primer and universal primer to carry out pcr amplification reaction;

(4) hybridization: the liquid that moves of cartridge inside was robbed and all products all are put into hybridization are detected groove and hybridize after pcr amplification was finished, and the well heater of chip bottom at 65 ℃, moves temperature control liquid and robs and do Rapid Cleaning during hybridization after hybridization in 15 minutes finishes;

(5) signal detection: the cartridge of having cleaned is put into the last signal detection of acceptance in the cartridge reading apparatus, there are proofing units such as laser confocal scanning system and photomultiplier cartridge reading apparatus the inside, the data of reading directly manifest with the form of numeral by the systems analysis software processes, cartridge is just taken out from reading apparatus and is directly thrown away, and closed system has been avoided the generation of polluting.