CN106932574A

CN106932574A - The detection method of Type B streptococcal infection

Info

Publication number: CN106932574A
Application number: CN201710125339.8A
Authority: CN
Inventors: 丁弘; 唐克健; 李铮; 李立和
Original assignee: Tianjin Baodi Hospital
Current assignee: Tianjin Baodi Hospital
Priority date: 2017-03-04
Filing date: 2017-03-04
Publication date: 2017-07-07
Anticipated expiration: 2037-03-04
Also published as: CN106932574B

Abstract

The invention discloses a kind of detection method of Type B streptococcal infection, belong to the method for determining internal blood characteristics.Kit of the invention built with detection negative control sera, positive control serum, concentrated phosphoric acid buffer solution, adsorbent, reagent and the bio-sheet materials such as goat-anti people's IgM globulin of marked by fluorescein isothiocyanate, mountant, bio-sheet material reaction zone endoperidium have Type B streptococcus.Assay method is：Add the dilute serum of removal IgG antibody in reaction zone, after being incubated, rinsing, dry, add goat-anti people's IgM globulin of marked by fluorescein isothiocyanate, goat-anti people's IgM globulin fluorescent compositions of marked by fluorescein isothiocyanate are formed around thalline, and yellow-green fluorescence is produced, the inventive method is above existing method in indexs such as sensitivity, specificity.

Description

The detection method of Type B streptococcal infection

Technical field

Detection method the present invention relates to measure internal blood characteristics, the specifically detection method of Type B streptococcal infection.

Background technology

Type B (B-mode) streptococcus (GBS) is a kind of common enteral bacterium, about the enteron aisle of 10-35% healthy womens or It is the trace that this bacterium can be found inside vagina, the puerpera of this Bacteria carrying status has any opportunity for fetus, neonate exposed to infection Source, so that nearly 1% neonate infects, thus brings neonate great life threat, if meeting the bacterium of fierce type Kind, the course of disease rapidly deteriorates and causes neonatal death, and it is often the chief culprit for causing infection of newborn, the urogenital system of pregnant woman Type B streptococcus is such as carried, although itself will not fall ill, child may be vertically transmitted to, seriously may be lethal, new life can be caused Youngster's infection, septicemia and neonatal meningitis, general premature labor, under-weight, early stage one's water breaks are all associated, in neonate's birth Breaking-out in one week after, and symptom rapidly deteriorates so that dead within 48 hours.Sufferer is mostly the infant of low-birth weight, and most A kind of fearful symptom is to show septic shock with respiratory distress.Can be advised preferably existing according to U.S.'s gynecological 35 to a 37 weeks beta streptococcus culture of work, if category is positive and first considering with following risk factor is treated really:Mother Mother's villous amniosis, mother's one's water breaks are more than the puerpera that 18 hours, all premature of the number less than 35 weeks of pregnancy give birth to a child, neonate's sense Dye Type B streptococcus, may result in meningitis, bacteremia, the death rate up to 5%, wherein, it is slow that meningitis can also leave intellectual development The sequelae such as slow.Infection can be divided to two kinds:Early onset infects, and incidence is 0.2%；Infected within after birth 7 days to 3 months. Delayed infection, incidence is 0.7% to 0.8%.Delayed onset neonatal B type streptococcal infection, current Pathogenic pathway is not clear, newly Raw youngster's infection Type B streptococcus, symptom includes the situations such as hypothermia, fever, baby's anorexia, energy be not good, or even meeting Baby is caused bacteremia, meningitis and pneumonia occur, wherein, the baby for having meningitis can leave sequelae, including regard Power, hearing it is slow by the intellectual development in future is undermined.In Type B streptococcus generally existing natural environment, female urinary is common in Reproductive system, the majority women that carries disease germs can't fall ill, and a few peoples have urethral infection symptom:Chilly, fever, uterus tenderness, Solution urine pain, urgent urination and pyuria.

Clinical labororatory's Type B streptococcal infection detection method is a lot, including bacterial cultivation, vaginal swab immunochromatography The assay methods such as method, Gold standard, vaginal swab PCR methods, some method assay method accuracys rate are high but time-consuming, and high cost has Method is easy, quickly, as a result relatively accurate, and anti-Type B streptococcus anti-IgM is pregnant to judge during present invention research one kind determines blood Whether woman infects the streptococcic method of Type B.

The content of the invention

One, box bodys

Kit of the invention is hexahedral shape, is made up of with cassette bottom lid, is characterised by cassette bottom near side long Any a line be provided with place reagent bottle bottle stand；Bottle stand is provided with multiple jacks for placing reagent bottle；The centre of cassette bottom One is provided with cassette bottom is divided into placement reagent and the biological two-part catch of slide glass is placed, including rectangle box body (1) and and its The lid (2) of one side edge bending disjunctor, it is characterised in that：Reagent rack (3) is set in box body (1), there are 5 same holes in reagent rack The jack (4) in footpath, is placed with adsorbent (9) in jack, goat-anti people IgM globulin (10) of marked by fluorescein isothiocyanate is negative Control serum (11), positive control serum (12), mountant (13), the centre of cassette bottom is provided with one and cassette bottom is divided into placement buffering Liquid reagent (8) and placement bio-sheet material (5) two-part papery catch (6), biological slide glass (5) outside plastics package (7).

Brief description of the drawings

Fig. 1 is the schematic diagram of kit of the present invention；

Fig. 2 is the schematic diagram of slide glass of the present invention.

1. the lid of box body 2.

3. the jack of reagent rack 4.

5. the papery catch of bio-sheet material 6.

7. the buffer solution of bio-sheet material plastics package 8.

9. goat-anti people's IgM globulin of the marked by fluorescein isothiocyanate of adsorbent 10.

The positive control serum of 11. negative control sera 12.

13. mountants

5a, 5b, 5c are test reaction area wherein on bio-sheet material (5).

The article that two, kits are equipped with

1 bottle of negative control sera, positive control serum, mountant, suction are respectively placed with the bottle stand 3 of kit of the present invention Attached dose, goat-anti people's IgM globulin of marked by fluorescein isothiocyanate, cassette bottom left half are placed with two bottles of phosphate of 50ml concentrations (containing 10.2 grams of buffered phosphate) buffer solution, right half is placed with 8 bio-sheet materials with packaging bag, and wherein adsorbent is sheep Anti-human IgG antibodies, can adsorb IgG in blood, and IgM is combined with bio-sheet material thalline in making blood.

3rd, the making of biological slide

The Type B streptococcus that the making application of bio-sheet material is separate carries out Zengjing Granule, is washed using phosphate buffer Wash 5 times, carry out 15000G centrifugation 10min, abandoning supernatant, separation of bacterial continues with 10ml PBS-T buffer solutions 5% (V/V) TritonX-100PBS is suspended, and cell number is adjusted to 100/μ l with PBS, spot film to containing on poly-D-lysine slide, cold wind After drying, sealing, -70 DEG C of Cord bloods.

Specific embodiment

Embodiment 1

1. after detection method takes bio-sheet material placement room temperature, by 25 μ L serum samples addition, 25 μ L PBS, incite somebody to action Serum after 30 μ L dilutions is added in 300 μ L adsorbents, is thoroughly mixed, and the serum after treatment wants 13000rpm centrifugations to remove for 10 minutes Precipitation is gone, yin and yang attribute control is not required to sorbent treatment；In the serum that the μ L sorbent treatments of every Kong Zhongjia 25 of slide are crossed, room temperature 60min, 0.01mol/L PBS wash 2min 3 times, and goat-anti people's IgM globulin of marked by fluorescein isothiocyanate, 20~37 is added dropwise DEG C 1~2h, 0.01mol/L PBS wash 2min 4 times；Mountant mounting, observes under 10 X of fluorescence microscope, 40 times of visuals field；Different sulphur Cyanic acid fluorescein is excited in 490~495nm, and fluorescence is launched in 520~530nm, such as it is positive be in yellow green, it is negative then occur without Yellow-green fluorescence, the method is simultaneously compared analysis with PCR method.

2.PCR methods carry out quantitative determination by B races streptococcus (GBS) DNA in vagina or rectal secretions, can be used for The auxiliary diagnosis of GBS infection and curative effect monitoring.Using PCR (PCR) technology combined with fluorescent probe technique, to GBS Nucleic acid specific fragment (CAMP) carries out fluorescence quantitative PCR detection, so as to judge the presence of GBS.The streptococcus nucleic acid determination examination of B races Agent box (fluorescent PCR method) is purchased from Xiamen Thymopetidum Injection thing Science and Technology Co., Ltd., and instrument is that the grand stone SLAN-96P in Shanghai is full-automatic Be added to the extract of 5 μ L samples, the positive, negative control in PCR reaction tubes by medical PCR analysis systems, assay method, instantaneously Centrifugation, is expanded by following amplification program：37 DEG C of 2min predegenerations；94℃2min；Then 94 DEG C of 20sec, 55 DEG C of 45sec elder generations Do 10 circulations, last 94 DEG C of 20sec；55 DEG C of 45sec, do 30 circulations.At 60 DEG C, reaction system is 50 for single-point fluoroscopic examination μL。

3. for a better understanding of the present invention, this is further illustrated below by the result drawn with two kinds of detection methods Good effect of the invention in terms of detection B races streptococcus：

3.1 research object in January, 2015 in January, 2016, in obstetrics of our hospital just before giving birth patient 58, at 20~35 years old age, puts down Equal 29.5 years old.

3.2B races streptococcus detection method：1. detection method；2.PCR test method(s)s.This two methods is respectively adopted Detection 58 B races streptococcus patients and control group, and two methods are carried out into statistical analysis.

The two methods testing result of table 1 compares

The two methods testing result of table 2 compares (%)

By table 1, table 2 method of testing of the invention can be seen in sensitivity, specificity, positive predictive value, negative predictive value Deng Indexes Comparison closely, two methods testing result coincidence rate is more than 80%, therefore, it can to carry out B using this method The detection of type streptococcal infection.

Claims

1. a kind of detection method of Type B streptococcal infection, it is characterised in that：After taking bio-sheet material placement room temperature, by 25 μ L serum Sample is added in 25 μ L PBS, and the serum after 30 μ L are diluted is added in 300 μ L adsorbents, is thoroughly mixed, the serum after treatment Want 13000rpm to be centrifuged and remove within 10 minutes precipitation, yin and yang attribute control is not required to sorbent treatment；In the μ L of every Kong Zhongjia 25 of slide The serum that sorbent treatment is crossed, room temperature 60min, 0.01mol/L PBS washes 2min 3 times, and marked by fluorescein isothiocyanate is added dropwise Goat-anti people's IgM globulin, 20~37 DEG C of 1~2h, 0.01mol/L PBS wash 2min 4 times；Mountant mounting, fluorescence microscope 10 Observed under 40 times of visuals field of X, fluorescein isothiocynate is excited in 490~495nm, fluorescence is launched in 520~530nm, be such as the positive It is negative then occur without yellow-green fluorescence in yellow green.

2. the detection method of a kind of Type B streptococcal infection according to claim 1, it is characterised in that mentioned reagent box includes Negative control sera, positive control serum, concentrated phosphoric acid buffer solution, adsorbent, the goat-anti people IgM of marked by fluorescein isothiocyanate Globulin, mountant.

3. the detection method of a kind of Type B streptococcal infection according to claim 1, it is characterised in that have on slide glass 3 it is anti- Area is answered, each reaction zone is coated with Type B streptococcus and is made bio-sheet material.