CN106932565A - TORCH detection kits and detection method - Google Patents
TORCH detection kits and detection method Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The present invention relates to a kind of TORCH detection kits and detection method.The detection kit includes microcarrier, immune response antibody and detection reagent.Microcarrier has coding information, and different for the coding information of the microcarrier of different TORCH pathogen, and micro-carrier surface is coated with TORCH pathogen.Immune response antibody includes anti-human IgG antibodies and anti-human IgM antibodies, and anti-human IgG antibodies and anti-human IgM antibodies are respectively connected with detection label.Detection reagent contains can be with the fluorescin of detection label reaction bonded.The detection kit can be used to detect up to six kinds IgG antibodies or IgM antibody of TORCH pathogen, such that it is able to enrich testing result, can more efficiently adjuvant clinical diagnosis.
Description
Technical field
The present invention relates to pathogen detection field, more particularly, to a kind of TORCH detection kits and detection side
Method.
Background technology
TORCH refers to the cause of disease that can cause congenital intrauterine infection and perinatal infection and cause peri-natal infant deformity
Body, it is one group of English name abbreviation of pathogenic microorganism.Wherein T (Toxoplasmosis gondii) is bow
Slurry worm；O (Others) represents other, including syphilis (Syphilis), varicella virus
(Varicella-zoster), HPV B19 (Parvovirus B19) etc.；R (Rubella virus) generations
Table rubella virus；C (Cytomegalovirus, CMV) represents cytomegalovirus；H(Herpes simplex
Virus, HSV) herpes simplex virus is represented, it is divided into the first type (Herpes simplex virus type 1, HSV1)
With Second-Type (Herpes simplex virus type 2, HSV2).TORCH can cause mother and baby to infect, pregnant woman
Because endocrine alteration and immunity degradation easily occur primary infection, virus can be by placenta or product during infection
Road transmission causes premature labor, miscarriage, stillborn foetus or monster to fetus, it is also possible to cause neonate's multisystem to damage
Evil, especially in the organogenetic period at pregnancy initial stage, destruction cell is possible to if being infected by the virus or suppresses thin
The division of born of the same parents and propagation, if in organogenetic period postoperative infection, tissue and organ may be destroyed, and if pregnant woman
Once infected, and hid and be also easily activated and produce recurrent infection in internal virus.
Earth's bow shock (Toxoplasmosis gondii) is distributed widely in nature, and main host is the ridge of homoiothermy
Vertebrate, but the parasitic selectivity of Earth's bow shock is not high, therefore people, pig, ox are all likely to become intermediate host,
And cat is the final host of Earth's bow shock, therefore its main infection approach is typically because eating by cat fecal pollution
Food is not cooked meat or soil by Earth's bow shock parasitism.Human infection's Earth's bow shock is not in generally obvious
Symptom and meeting lifetime immunity.If but pregnant woman, in gestation primary infection, possible vertical infection fetus is made
Into congenital Earth's bow shock disease, common sympton is choroidoretinitis, intracranial calcification and water brain disease, causes tire
Youngster is lopsided, cause the sequelae such as miscarriage and neonatal disorder.
Rubella virus (Rubella virus, also known as german measles virus) is a kind of acute virus of highly infective,
Clinical symptoms are usually feveret, tired, mild rhinitis and with general irregular papule, typically in
Retroauricular lymph nodes, occipital bone inferior gluteal lymph node and after neck enlargement of lymph nodes be common symptom, but period of disease is not long
And symptom is gentle.If period of gestation infects german measles virus, can pass through placenta vertical infection and made to fetus
Impaired into dead, miscarriage or major organs, in 12 weeks interior the infecteds of first half period of gestation, fetus has more than 25% machine
Rate produces congenital German measles syndrome, and in the infected in 16 weeks, it is single that fetus then has 10% probability to produce
One birth defects, if just being infected in after 20 weeks, give birth to deformed child probability then very little.
Cytomegalovirus (Cytomegalovirus, CMV) is a kind of common virus, belongs to human herpes
Virus, can hide and lifelong band is former, and initial infection does not have manifest symptom, therefore most people will not be realized certainly
Oneself infects cytomegalovirus.And cytomegalovirus infection is the most common cause of disease of congenital infection disease, energy
Fetus, baby is caused seriously to damage or dead, wherein causing central nervous system sequelae most serious of all.]
The clinical manifestation for infecting cytomegalovirus has：
(1) congenital infection, pregnant woman can cause the congenital infection of fetus in infection in 12 weeks gestational periods,
For subclinical infection or stillborn foetus, miscarriage, premature labor and congenital abnormality may be caused；
(2) infection of newborn, be born 12 weeks in infect occur pneumonia, hepatitis, enlargement of lymph nodes and
Fash etc.；
(3) children and adult infect, majority be subclinical infection, minority occur monocytosis,mononucleosis,
Pneumonia, hepatitis and myocarditis；
(4) immune deficiency and organ transplant patients are infected, and performance each organ infection of whole body may be caused to cause
Dead rate is high.
Herpes simplex virus (Herpes simplex virus, HSV) is a kind of disease of viral communication, infection
Still there is the possibility of recurrence after having local primary focus, but recovery from illness afterwards, and be divided into the first type and Second-Type.
The type of herpes simplex virus first (HSV1), it is multiple to be born in child, but most of primary infection symptom
It is gentle unobvious, if infection may produce lethal congenital herpes simplex to neonate.And this kind of
The infection of primary and recidivity may all have influence on central nervous system, because antiviral therapy can reduce death
Rate, therefore the test alive that can do brain tissue is used to early diagnose suspected case.
Herpes simplex virus Second-Type (HSV2), this kind of virus generally causes genital herpes, if pregnancy woman
This is viral for woman's vagina infection, then have danger very high to be transmitted to neonate, causes neonate's internal organ more
The infection of unrestrained property, even encephalitis, death, while HSV2 is also the risk factor of uterus neck cancer.
B19 piconaviruses are the common Rash dieases of child, are the erythema infectioum cause of disease, if adult's infection,
Symptom is general slight only to feel arthralgia or swelling, if but infection of pregnant women to be then likely to result in fetus dead
Die, additionally, severe anemia occurs in the sufferer that the virus is also possible to cause immunity low.B19 piconaviruses are not only
It is the common Rash diease cause of disease of paediatrics, can also causes chronic hemolysis sufferer that acute Polyarthropathy occurs, with
And cause the infection of immunodeficiency sufferer continuation.If in First Trimester the infected, being likely to result in fetus palace
Interior anaemia, fetus edema, heart malformations etc. injure, if but in infected after 20 weeks on fetus just without influence.
At present detection TORCH mainly use immunological method, by infection human body after produce IgG or
IgM is judged, verifies that specific IgG is represented and infected, and has infected one period, if verifying
Specific IgM is then expressed as recent infection, or hides and be activated and produce recurrent infection in internal virus
Possibility, but available reagent group still be generally it is single detection or can simultaneously check project limited, it is impossible to immediately provide
Complete survey result effectively assists clinical diagnosis.
Prior art is prevalent in problems with and waits to improve：
(1) the sample to be tested demand of single detection reagent group is big, and collection sample to be tested causes sufferer to bear
Load；
(2) commercially available multivariate detection reagent set and method, it is possible to provide disposable detection project is limited, can drop
Low sample requirement but cannot effectively lift checkability；
(3) test results report is only capable of providing sufferer inspection data with analysis, it is impossible to while providing inspection
Reagent set complete information is as reference.
The content of the invention
Based on this, it is necessary to provide TORCH detection reagents a kind of low to sample requirements and that detection efficiency is high
Box and detection method.
A kind of TORCH detection kits, including microcarrier reagent, immune response antibody and detection reagent；
Containing the microcarrier for being coated with TORCH pathogen in the microcarrier reagent, and for different
The coding information of the microcarrier of TORCH pathogen is different；The microcarrier reagent contains to be coated with to bend to be starched
The microcarrier of worm, the microcarrier for being coated with rubella virus, the microcarrier for being coated with cytomegalovirus, it is coated with
The microcarrier of the type of herpes simplex virus first, the microcarrier and coating that are coated with herpes simplex virus Second-Type
There is at least one in the microcarrier of B19 piconaviruses；
The immune response antibody includes anti-human IgG antibodies and anti-human IgM antibodies, and the anti-human igg is anti-
Body and anti-human IgM antibodies are respectively connected with detection label；
The detection reagent contains can be with the fluorescin of the detection label reaction bonded.
Wherein in one embodiment, the microcarrier reagent contains microcarrier, the coating for being coated with Earth's bow shock
The microcarrier that has rubella virus, the microcarrier for being coated with cytomegalovirus, it is coated with herpes simplex virus first
The microcarrier of type, the microcarrier for being coated with herpes simplex virus Second-Type and it is coated with B19 piconaviruses
Microcarrier.
Wherein in one embodiment, the amino that the antigen coat thing of the micro-carrier surface is carried by it with
The micro-carrier surface is coated on after the activated carboxyl reaction of the corresponding micro-carrier surface.
Wherein in one embodiment, the anti-human IgG antibodies are goat anti-human igg antibody, the anti-human IgM
Antibody is goat-anti human IgM antibody.
Wherein in one embodiment, the detection label is biotin, and the fluorescin is strepto- parent
With the algae red fluorescin of element mark.
Wherein in one embodiment, also including pre-treatment reagent, the pre-treatment reagent has two groups, wherein
One group is detected for IgG antibody, and another group is detected for IgM antibody；
For IgG antibody detection pre-treatment reagent contain the BSA of 10mg/mL, 1 × PBS,
The NaN of 0.05wt%3, 1wt% PVA and deionized water；
For IgM antibody detection pre-treatment reagent contain goat anti-human igg antibody Fc that volumetric concentration is 50%,
0.025wt%NaN3, 0.5 × PBS, 1wt%PVA and deionized water.
Wherein in one embodiment, also including positive quality control product, negative quality-control product and six kinds of threshold value quality-control products.
A kind of TORCH detection methods, using the TORCH detection kits described in any of the above-described embodiment,
The TORCH detection methods comprise the following steps：
The sample to be tested is divided into two groups, one group is detected for IgG antibody, another group is used for IgM antibody
Detection, sample to be tested is added in the corresponding micropore；
Microcarrier reagent is added in the micropore, in 30-50 DEG C, 500-2000rpm concussion reactions；
After cleaning the porous plate, the reagent containing the immune response antibody is added in the micropore, in
30-50 DEG C, 500-200rpm concussion reactions, wherein, the group for IgG antibody detection adds anti-human igg to resist
Body, the group for IgM antibody detection adds anti-human IgM antibodies；
The detection reagent is added in the micropore, in 30-50 DEG C, 500-2000rpm concussion reactions；
After cleaning the porous plate, the coding information of the microcarrier is recognized using image recognition system, and examined
Corresponding marking signal is surveyed, testing result is obtained through analyzing and processing.
Wherein in one embodiment, before being additionally included in addition sample to be tested, added in corresponding micropore
Pre-treatment reagent, and after the sample to be tested is added, by the pre-treatment reagent and the sample to be tested in
30-50 DEG C, 500-2000rpm concussion reactions the step of.
Wherein in one embodiment, also including to addition positive quality control product, feminine gender in the different micropores
The step of quality-control product and six kinds of threshold value quality-control products, to the positive quality control product, negative quality-control product and six kinds of threshold values
The treatment of quality-control product is with the sample to be tested.
Wherein in one embodiment, the detection label is biotin, and the fluorescent marker is strepto-
The algae red fluorescin of Avidin mark；It is described to detect that corresponding marking signal is to use fluorescent brightness detection group
Part is detected and quantifies the fluorescence signal on the microcarrier.
Wherein in one embodiment, the image recognition system is DigiPlex Analyzer；At the analysis
Reason is to be analyzed treatment using the software systems DeXipher of DigiPlex Analyzer.
TORCH detection kits of the invention and its detection method have the advantages that：
(1) detection kit can detect simultaneously the corresponding IgG antibody of up to six kinds TORCH pathogen or
IgM antibody, such that it is able to enrich testing result, can more efficiently adjuvant clinical diagnosis；
(2) when microcarrier, coated antibody and detection antibody have various in detection kit, the detection reagent
Box can simultaneously detect various mankind TORCH pathogen, without repeated detection, can reduce required sample size, and
The accuracy of detection efficiency and result can be effectively improved；
(3) multivariate detection can increase clinic with multiple screening and with reference to diversification biomedicine detecting system
Precision and simplicity；
(4) image recognition system combination software analysis can obtain a large amount of and complete testing results and believe with reagent set
Breath, convenient data processing, object information is comprehensive.
Brief description of the drawings
Fig. 1 is the experiment flow figure of embodiment 1.
Specific embodiment
For the ease of understanding the present invention, the present invention is described more fully below with reference to relevant drawings.
Presently preferred embodiments of the present invention is given in accompanying drawing.But, the present invention can come real in many different forms
It is existing, however it is not limited to embodiment described herein.On the contrary, the purpose for providing these embodiments is made to this
The understanding of disclosure of the invention content is more thorough comprehensive.
The TORCH detection kits of one implementation method, including microcarrier reagent, immune response antibody and detection
Reagent.
Contain microcarrier (Microcarrier) in microcarrier reagent.Microcarrier is made using magnetic material, is had
Magnetic.Microcarrier is provided with and is provided with and repaiies for the coding information for image recognition system identification, and its surface
Decorations layer, such as physical modification layer or chemical modification layer, preferably chemical modification layer.Wherein chemical modification layer can with but
It is not limited to the decorative layer formed via carboxylation treatment.Can be by for the microcarrier that is processed through carboxylation
NHS (N-Hydroxysuccinimide, N-hydroxy-succinamide) and EDC
(1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, 1- (3- dimethylaminopropyls) -3- ethyls
Carbodiimide) activated carboxyl reacted, and will carry the antigen binding of amino in micro-carrier surface, forms anti-
The microcarrier of primordial covering.
Microcarrier capacity is high, can be used as multiple vectors.When detection multiple pathogens are required to, microcarrier
Can targetedly be designed as it is various, to be coated with different antigen coat things.In the present embodiment, micro- load
Body is at least a kind of, for six kinds of TORCH pathogen：Earth's bow shock, rubella virus, cytomegalovirus, list
At least one design in the type of pure herpesviral first, herpes simplex virus Second-Type and B19 piconaviruses.
Coding information on the microcarrier of the different TORCH pathogen of coating is different.
Antigen coat is in micro-carrier surface.Antigen can be connected with microcarrier by physical action or chemical action.
Connected for chemical action, capture group can be connected on antigen.Capture group and micro-carrier surface
Chemical modification layer correspondence, can be with the chemical modification of micro-carrier surface layer covalent bond.In one embodiment,
Capture group can be amino etc., and amino can be with the activated carboxyl reaction bonded of micro-carrier surface.
The microcarrier of antigen coat can be used but be not limited to following method and prepare：
After microcarrier is cleaned with the 100mM MES reagents containing 15%EtOH, sequentially add
In the 100mM MES of 15%EtOH, reaction condition is 50mg/mL NHS and 50mg/mL EDC
Room temperature rotation concussion 20 minutes, this reaction is activation micro-carrier surface.Added after cleaning and be intended to be coated with what is used
Antigen, reaction condition is room temperature rotation concussion two hours, and this reaction is conjugation, is added after cleaning and contained
The TBS of 1%BSA, reaction condition is room temperature rotation concussion 30 minutes, and this reaction is the preposition reaction of affinity,
Treat that above reaction is completed, i.e. completion coating process is finally cleaned with PBST.
In the present embodiment, microcarrier reagent contains and is coated with the microcarrier of Earth's bow shock, is coated with rubella
Poison microcarrier, be coated with the microcarrier of cytomegalovirus, be coated with micro- load of the type of herpes simplex virus first
Body, it is coated with the microcarrier of herpes simplex virus Second-Type and is coated with the microcarrier of B19 piconaviruses
At least one.Preferably, microcarrier reagent contains above-mentioned six kinds of microcarriers, namely six kinds of microcarriers are mixed
It is combined and is made mix reagent, the mix reagent can be used for while detecting above-mentioned six kinds of TORCH pathogen.
In the present embodiment, microcarrier reagent has two groups, and one group for IgG antibody detection, another group of use
In IgM antibody detection.For same TORCH pathogen, for the microcarrier of its IgG antibody detection
Can be with identical with coated antigen on the microcarrier detected for its IgM antibody, it is also possible to different, such as one
In implementation method, for IgG antibody detection microcarrier reagent and for IgM antibody detect microcarrier reagent
In the coated antigen of microcarrier can select it is as shown in table 1 below：
Table 1
Immune response antibody includes anti-human IgG antibodies and anti-human IgM antibodies.Anti-human IgG antibodies and anti-human IgM
Antibody is respectively connected with detection label.In the present embodiment, anti-human IgG antibodies are goat anti-human igg antibody
(goat anti-human IgG antibody), anti-human IgM antibodies are goat-anti human IgM antibody (goat
anti-human IgM antibody).Two kinds of immune response antibody are prepared into immune response reagent respectively, with respectively
For the detection of IgG antibody and IgM antibody in test serum equal samples.As in one embodiment, it is immunized
Reagin can select as shown in table 2 below：
Table 2
Detection reagent contains can be with the fluorescin of detection label reaction bonded.In the present embodiment,
Detection label is biotin, and fluorescin is the algae red fluorescin of marked by streptavidin.It is understood that
In other embodiments, detection label and fluorescin are not limited to described above, and such as detection label can be with
It is Streptavidin, fluorescin can be fluorescin of Avidin mark etc..
Additionally, in the present embodiment, the detection kit also include pre-treatment reagent, positive quality control product,
Negative quality-control product and six kinds of threshold value quality-control products.
Pre-treatment reagent can reduce background-influence.In the present embodiment, pre-treatment reagent has two groups,
One of which detects that another group is detected for IgM antibody for IgG antibody.For IgG antibody detection
Pre-treatment reagent contains the BSA of 10mg/mL, 1 × PBS, NaN3,1wt% of 0.05wt%
PVA and deionized water.Pre-treatment reagent for IgM antibody detection contains the sheep that volumetric concentration is 50%
Anti-human IgG antibodies Fc, 0.025wt%NaN3,0.5 × PBS, 1wt%PVA and deionized water,
The catalog number of EQUITECH-BIO companies can such as be selected for the reagent of GAHGFC-0500.Specifically
It is as shown in table 3 below：
Table 3
Positive quality control product, negative quality-control product and threshold value quality-control product have each affiliated micropore when being detected,
Will not mix with sample to be tested, but together must carry out complete experiment flow with sample to be tested and obtain respective
MFI values, if desired for being reacted with pre-treatment reagent and microcarrier reagent reacting and contain immune response antibody
Reagent reacting and with detection reagent reaction etc..Its positives MFI value with negative quality-control product can be used as treating
Test sample sheet or experiment flow have the judgment basis of no problem, and the MFI values of threshold value quality-control product can then provide software
Default judgment principle carries out sample analysis, i.e. S/CO values, and wherein S represents the Signal values of sample to be tested,
CO (CutOff) represents threshold value, and gained S/CO >=1.1 represent positive (Positive), and S/CO ＜ 0.9 represent negative
(Negative), 0.9≤S/CO ＜ 1.1 are then represented uncertain (Indeterminate).
In the present embodiment, threshold value quality-control product can select the serum plasma bought from Cerba, can basis
Known results, pick out and contain Earth's bow shock, rubella virus, cytomegalovirus, herpes simplex virus first respectively
Type, herpes simplex virus Second-Type and B19 piconaviruses are used as threshold in six kinds of serum plasmas of weakly positive
Value quality-control product.Then according to the threshold value of gained is carried out with a collection of experiment as secondary standard is worked as when using, such as substantially
Test samples more than the threshold value (S/CO >=1.1) are then judged as the positive, otherwise are then judged as feminine gender (S/CO
＜ 0.9), (0.9≤S/CO ＜'s 1.1) close with the threshold value is expressed as uncertain sample, it is necessary to reference to other
Testing inspection.
Positive quality control product can select the serum plasma bought from Cerba, can be picked out according to known results
Containing Earth's bow shock, rubella virus, cytomegalovirus, the type of herpes simplex virus first, herpes simplex virus second
Type and the positive serum plasma of B19 piconaviruses display (wherein also may same pipe serum to be mixed
Blood plasma contains more than one pathogen), then according to experimental result, its MFI value must be substantially big for its mixed proportion
Selected threshold value quality-control product in same batch is selection standard.
Negative quality-control product can select 100% Normal Goat Serum (Normal Goat Serum), such as Jackson
The Normal Goat Serum of the catalog number 005-00-121 of ImmunoResearch productions.
Present embodiment additionally provides a kind of TORCH detection methods, and it uses above-mentioned TORCH detection reagents
Box.The TORCH detection methods comprise the following steps：
Step S110, pre-treatment reagent is added in the micropore of the pending reaction of porous disc, is carried on the back for reducing
The influence of scape value.
The addition of pre-treatment reagent is divided into two groups of additions, for IgG antibody detection pre-treatment reagent and be used for
The pre-treatment reagent of IgM antibody detection is added separately in different micropores.
Step S120, two groups are also classified into by sample to be tested, and one group is detected that another group is used for for IgG antibody
IgM antibody detection, sample to be tested is added in corresponding micropore, is shaken in 30-50 DEG C, 500-2000rpm
Reaction.
In the present embodiment, also including to added in different micropores positive quality control product, negative quality-control product and
The step of six kinds of threshold value quality-control products, to positive quality control product, negative quality-control product and six kinds for the treatment of of threshold value quality-control product
Same sample to be tested.
Step S130, adds microcarrier reagent, in 30-50 DEG C, 500-2000rpm concussion reactions in micropore.
Step S140, after cleaning porous plate, adds the reagent containing immune response antibody in micropore, in
30-50 DEG C, 500-200rpm concussion reactions, wherein, the group for IgG antibody detection adds anti-human igg to resist
Body, the group for IgM antibody detection adds anti-human IgM antibodies.
In the present embodiment, when cleaning porous plate, porous plate is placed on magnetic devices, to adsorb tool
Magnetic microcarrier, is adsorbed the bottom hole in micropore, below similarly.
Step S150, adds detection reagent in micropore, in 30-50 DEG C, 500-2000rpm concussion reactions.
Step S160, after cleaning porous plate, the coding information of microcarrier is recognized using image recognition system, and
Corresponding marking signal is detected, testing result is obtained through analyzing and processing.
Label used by present embodiment is the algae red fluorescin reagent of marked by streptavidin, and it is right to detect
The marking signal answered is to be detected using fluorescent brightness detection components and quantify the fluorescence signal on microcarrier, including
Image recognition system and fluorescent brightness detection components, with reference to CCD camera and the product of fluorescence microscope.This reality
The image recognition system used by mode is applied for DigiPlex Analyzer, including image recognition system and fluorescent brightness
Detecting element, can be placed in porous plate in DigiPlex Analyzer and be read out and divide by user after the completion of reaction
Analysis, the instrument can take pictures in selected region, and be picked out in image on each microcarrier by image recognition system
Coding information.Analyzing and processing is to be analyzed treatment using the software systems DeXipher of PlexBio100,
Because each antigen is corresponding with the coding information on microcarrier when being coated with, the condition is also previously written software system,
So system can successfully pick out the corresponding signal of each microcarrier.Related letter is exported after being calculated through software analysis
Breath, including:The information such as reagent name, reagent lot, operator, the TORCH pathogen of microcarrier correspondence
And testing result.
TORCH detection kits of the invention and its detection method have the advantages that：
(1) detection kit can detect simultaneously the corresponding IgG antibody of up to six kinds TORCH pathogen or
IgM antibody, such that it is able to enrich testing result, can more efficiently adjuvant clinical diagnosis；
(2) when microcarrier, coated antibody and detection antibody have various in detection kit, the detection reagent
Box can simultaneously detect various TORCH pathogen, without repeated detection, can reduce required sample size, and can have
Effect improves the accuracy of detection efficiency and result；
(3) multivariate detection can increase clinic with multiple screening and with reference to diversification biomedicine detecting system
Precision and simplicity；
(4) image recognition system combination software analysis can obtain a large amount of and complete testing results and believe with reagent set
Breath, convenient data processing, object information is comprehensive.
It is below specific embodiment part：
Embodiment 1
The detection kit of embodiment 1 can detect simultaneously the corresponding IgG antibody of six kinds of TORCH pathogen and
IgM antibody.Wherein, six kinds of TORCH pathogen be respectively Earth's bow shock, rubella virus, cytomegalovirus,
The type of herpes simplex virus first, herpes simplex virus Second-Type and B19 piconaviruses.Accordingly, every group
Contain six kinds of corresponding microcarriers in microcarrier reagent, for being coated with the microcarrier group of IgG antibody detection
Corresponding IgG antigens, for being coated with corresponding IgM antigens on IgM antibody detection microcarrier, specifically
IgG antigens and IgM antigens are as shown in Table 1 above.
It is understood that in other embodiments, the detection kit can be containing for detecting above-mentioned six kinds
One kind, two kinds, three kinds, four kinds or five kinds of corresponding microcarrier reagents and quality-control product in TORCH pathogen.
The detection method comprises the following steps：
The temperature that temperature incubates oscillator is set between 30-50 DEG C, is preheated.
100 μ L pre-treatment reagents are added in the micropore of the pending reaction of disposable 96 orifice plate, for reducing
Background-influence.The addition of pre-treatment reagent (as shown in Table 3 above) is divided into two groups of additions, for IgG
The pre-treatment reagent of antibody test and the pre-treatment reagent for IgM antibody detection are added separately to different micro-
Kong Zhong.
The human serum sample to be tested of collection is divided into two groups, one group for IgG antibody detection, another group of use
In IgM antibody detection, sample to be tested is added in corresponding micropore, and added in other corresponding micropores
Positive quality control product, negative quality-control product and six kinds of threshold value quality-control products, shake anti-in 30-50 DEG C, 500-2000rpm
Answer 15min.Sample to be tested is 5 μ L with the addition of quality-control product.
20 μ L microcarrier reagents are added in micropore, in 30-50 DEG C, 500-2000rpm concussion reactions 30min.
Wherein, the group for IgG antibody detection adds the microcarrier for being coated with IgG antigens, for IgM antibody inspection
The group of survey adds the microcarrier for being coated with IgM antigens.
After cleaning disposable 96 orifice plate, the reagent containing 50 μ L immune response antibody is added in micropore (such as
Shown in above-mentioned table 2), in 30-50 DEG C, 500-200rpm concussion reaction 30min, wherein, it is anti-for IgG
The group that physical examination is surveyed adds anti-human IgG antibodies, and the group for IgM antibody detection adds anti-human IgM antibodies.
50 μ L detection reagents are added in micropore, in 30-50 DEG C, 500-2000rpm concussion reactions 10min.
After cleaning disposable 96 orifice plate, disposable 96 microwell plate for completing will be operated to be put into image recognition system
DigiPlex Analyzer recognize microcarrier, and fluorescent brightness detecting element is analyzed and quantified glimmering on microcarrier
Light signal, after being finally analyzed calculating via the software system DeXipher of DigiPlex Analyzer, obtains
Report and testing result to testing reagent group information.Result is as shown in table 4 below and table 5.
Table 4
Note：Detection sample used by embodiment 1 is the human serum of known results, it is therefore intended that prove to use
TORCH kits of the invention and detection method can detect TORCH IgG and IgM really, such as correspondence
Contain corresponding antibody, second mankind in every kind of TORCH pathogen, wherein first man class serum sample
Corresponding antibody is not contained in serum sample, below similarly；
S represents that Signal, CO represent CutOff, is measured sample for the decision method of sample results
Divided by the threshold value MFI of the detection project, gained S/CO >=1.1 represent that Positive, S/CO ＜ 0.9 are represented to MFI
Negative, 0.9≤S/CO ＜ 1.1 then represents Indeterminate, similarly hereinafter.
Table 5
By above-mentioned table 4 and table 5 as can be seen that the TORCH detection kits can disposably for containing various
The sample to be tested of TORCH pathogen is detected, such that it is able to reduce required sample size, and effectively improved
Detection efficiency, and due to being disposable detection, without repeatedly sample-adding, result of the test is more accurate.The detection
Kit and detection method can combine multivariate detection with multiple screening and combine polynary metaplasia doctor's detection system
System, such that it is able to increase clinical precision and simplicity.And image recognition system and corresponding software can be combined
Analysis, can obtain a large amount of and complete testing results, and detection efficiency is further improved.
Each technical characteristic of embodiment described above can be combined arbitrarily, not right to make description succinct
The all possible combination of each technical characteristic in above-described embodiment is all described, as long as however, these skills
The combination of art feature does not exist contradiction, is all considered to be the scope of this specification record.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed,
But can not therefore be construed as limiting the scope of the patent.It should be pointed out that for this area
For those of ordinary skill, without departing from the inventive concept of the premise, some deformations can also be made and changed
Enter, these belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended power
Profit requires to be defined.
Claims (12)
1. a kind of TORCH detection kits, it is characterised in that resist including microcarrier reagent, immune response
Body and detection reagent；
Containing the microcarrier for being coated with TORCH pathogen in the microcarrier reagent, and for different
The coding information of the microcarrier of TORCH pathogen is different；The microcarrier reagent contains to be coated with to bend to be starched
The microcarrier of worm, the microcarrier for being coated with rubella virus, the microcarrier for being coated with cytomegalovirus, it is coated with
The microcarrier of the type of herpes simplex virus first, the microcarrier and coating that are coated with herpes simplex virus Second-Type
There is at least one in the microcarrier of B19 piconaviruses；
The immune response antibody includes anti-human IgG antibodies and anti-human IgM antibodies, and the anti-human igg is anti-
Body and anti-human IgM antibodies are respectively connected with detection label；
The detection reagent contains can be with the fluorescin of the detection label reaction bonded.
2. TORCH detection kits as claimed in claim 1, it is characterised in that the microcarrier examination
Agent contains the microcarrier for being coated with Earth's bow shock, the microcarrier for being coated with rubella virus, is coated with cytomegalovirus
Microcarrier, be coated with the microcarrier of the type of herpes simplex virus first, be coated with herpes simplex virus Second-Type
Microcarrier and be coated with the microcarrier of B19 piconaviruses.
3. TORCH detection kits as claimed in claim 1, it is characterised in that the microcarrier table
The antigen coat thing in face is after its amino for carrying reacts with the activated carboxyl of the corresponding micro-carrier surface
It is coated on the micro-carrier surface.
4. TORCH detection kits as claimed in claim 1, it is characterised in that the anti-human igg
Antibody is goat anti-human igg antibody, and the anti-human IgM antibodies are goat-anti human IgM antibody.
5. TORCH detection kits as described in claim 1 or 4, it is characterised in that the detection
Label is biotin, and the fluorescin is the algae red fluorescin of marked by streptavidin.
6. TORCH detection kits as claimed in claim 1, it is characterised in that also including pre-treatment
Reagent, the pre-treatment reagent has two groups, and one of which is detected for IgG antibody, and another group is used for IgM
Antibody test；
For IgG antibody detection pre-treatment reagent contain the BSA of 10mg/mL, 1 × PBS,
The NaN of 0.05wt%3, 1wt% PVA and deionized water；
For IgM antibody detection pre-treatment reagent contain goat anti-human igg antibody Fc that volumetric concentration is 50%,
0.025wt%NaN3, 0.5 × PBS, 1wt%PVA and deionized water.
7. TORCH detection kits as claimed in claim 1, it is characterised in that also including positive matter
Control product, negative quality-control product and six kinds of threshold value quality-control products.
8. a kind of TORCH detection methods, it is characterised in that using such as any one of claim 1-7 institutes
The TORCH detection kits stated, the TORCH detection methods comprise the following steps：
The sample to be tested is divided into two groups, one group is detected for IgG antibody, another group is used for IgM antibody
Detection, sample to be tested is added in the corresponding micropore；
Microcarrier reagent is added in the micropore, in 30-50 DEG C, 500-2000rpm concussion reactions；
After cleaning the porous plate, the reagent containing the immune response antibody is added in the micropore, in
30-50 DEG C, 500-200rpm concussion reactions, wherein, the group for IgG antibody detection adds anti-human igg to resist
Body, the group for IgM antibody detection adds anti-human IgM antibodies；
The detection reagent is added in the micropore, in 30-50 DEG C, 500-2000rpm concussion reactions；
After cleaning the porous plate, the coding information of the microcarrier is recognized using image recognition system, and examined
Corresponding marking signal is surveyed, testing result is obtained through analyzing and processing.
9. TORCH detection methods as claimed in claim 8, it is characterised in that be additionally included in addition and treat
Before test sample sheet, pre-treatment reagent is added in the corresponding micropore, and after the sample to be tested is added,
By the pre-treatment reagent and the sample to be tested in 30-50 DEG C, the 500-2000rpm concussion reactions the step of.
10. TORCH detection methods as claimed in claim 8, it is characterised in that also including to different
The step of positive quality control product, negative quality-control product and six kinds of threshold value quality-control products are added in the micropore, to the sun
Property quality-control product, negative quality-control product and six kinds for the treatment of of threshold value quality-control product with the sample to be tested.
The 11. TORCH detection methods as any one of claim 8-10, it is characterised in that institute
It is biotin to state detection label, and the fluorescent marker is the algae red fluorescin of marked by streptavidin；
It is described to detect that corresponding marking signal is detected and quantified on the microcarrier using fluorescent brightness detection components
Fluorescence signal.
The 12. TORCH detection methods as any one of claim 8-10, it is characterised in that institute
Image recognition system is stated for DigiPlex Analyzer；The analyzing and processing is to use DigiPlex Analyzer
Software systems DeXipher is analyzed treatment.
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| CN109116031A (en) * | 2018-07-25 | 2019-01-01 | 滴准生物科技（常州）有限公司 | A kind of detection kit can detect a variety of prenatal and postnatal care pathogen IgG antibodies |
| CN109001468A (en) * | 2018-08-06 | 2018-12-14 | 滴准生物科技（常州）有限公司 | A kind of detection kit of CMV antibody IgM |
| CN109270279A (en) * | 2018-10-18 | 2019-01-25 | 郑州安图生物工程股份有限公司 | TORCH-IgG antibody mixing quality-control product |
| WO2021134304A1 (en) * | 2019-12-30 | 2021-07-08 | 深圳迈瑞生物医疗电子股份有限公司 | Kit, method and immunoassay analyzer for screening torch infection |
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