阅读说明：本技术 一种人体血清叶酸的测定方法 (A kind of measuring method of human serum folic acid ) 是由 唐静 周义正 李漂 陈星星 于 2019-09-12 设计创作，主要内容包括：本发明公开了一种人体血清叶酸的测定方法,包括实验用水的制备、叶酸标准液配制、鼠李糖乳杆菌生长培养基配制、鼠李糖乳杆菌的制备、检测培养基配制、血清叶酸检测；本发明采用活性炭吸附水中的有机杂质,可以达到消除干扰的目的；叶酸的化学性质不稳定,容易被氧化,在生长和检测培养基中添加抗坏血酸作为抗氧化剂,使溶血液中的氧分尽量降低,从而增加脱氧血红蛋白的含量,达到保护叶酸的作用；检测时细胞培养板用封口膜封闭,有助于鼠李糖乳杆菌的生长,也能防止水分蒸发和叶酸的氧化；本发明简便易行、价格低廉、且灵敏度高,更适合大规模人群的叶酸筛查需要。(The invention discloses a kind of measuring methods of human serum folic acid, and preparation, folic acid titer including experimental water are prepared, Lactobacillus rhamnosus growth medium is prepared, the preparation of Lactobacillus rhamnosus, detection culture medium is prepared, serum folic acid detects；The present invention can achieve the purpose for eliminating interference using the organic impurities in activated carbon adsorption water；The unstable chemcial property of folic acid, is oxidized easily, and adds ascorbic acid in culture medium as antioxidant growing and detecting, reduces the oxygen in hemolysate as far as possible, to increase the content of deoxyhemoglobin, has the function that protect folic acid；Tissue culture plate is closed with sealed membrane when detection, facilitates the growth of Lactobacillus rhamnosus, can also prevent the oxidation of moisture evaporation and folic acid；Simple and easy to do, cheap and high sensitivity of the invention, is more suitable for the folic acid screening needs of large-scale crowd.)

1. a kind of measuring method of human serum folic acid, which comprises the steps of:

(1) preparation of experimental water: being added active carbon powder, stir evenly in deionized water, filter removal active carbon powder, obtain To experimental water；

(2) folic acid titer is prepared: being weighed folic acid standard items, is prepared the mark of various concentration surely with ammonia solvent and with experimental water Quasi- product solution；

(3) Lactobacillus rhamnosus growth medium is prepared: being weighed folic acid culture medium dry powder, antioxidant, chloramphenicol, manganese sulfate and is put Enter in beaker, adds folic acid standard solution and experimental water to be calibrated, be heated to boiling after mixing, then high pressure sterilization, obtain Growth medium dispenses after cooling, and -20 DEG C freeze, the preparation for strain；

(4) preparation of Lactobacillus rhamnosus: Lactobacillus rhamnosus is inoculated in growth medium, culture in 37 DEG C of incubators For 24 hours, 2000r/min is centrifuged 2-3min, discards supernatant liquid, and growth medium is added, is centrifuged 2-3min after mixing, discards supernatant Growth medium is added in liquid, and for 24 hours, logarithmic growth phase bacterium solution and 80% glycerol are mixed in 1:1 ratio for culture in 37 DEG C of incubators It closes, 2mL cryovial is sub-packed in after mixing, -20 DEG C freeze；

(5) detection culture medium is prepared: being weighed folic acid culture medium dry powder, chloramphenicol, manganese sulfate and is put into beaker, experiment is added and uses Water is heated to boiling, and antioxidant, Lactobacillus rhamnosus solution is added after cooling, mixes；

(6) serum folic acid detects: serum antioxidant being diluted 20 times in tissue culture plate, it is molten to be separately added into folic acid standard Liquid, blank well add the sodium azide solution that the standard solution that concentration is 0, concentration are 3%, and sample well adds the serum to be checked after dilution, Detection culture medium is added into all detection holes, sealed membrane blocks tissue culture plate, tissue culture plate is set 37 DEG C of incubation 36h Afterwards, it mixes, tears sealed membrane, stand 5min in microplate reader 590nm turbidimetric assay and select suitable standard curve fit mode Calculate the folic acid concentration of serum to be checked.

2. a kind of measuring method of human serum folic acid according to claim 1, which is characterized in that folic acid standard solution Concentration is 0.0,0.1,0.2,0.3,0.4,0.5 and 0.6ug/L of difference.

3. a kind of measuring method of human serum folic acid according to claim 1, which is characterized in that the antioxidant is Sodium ascorbate or propylgallate.

Technical field

The present invention relates to in-vitro diagnosis fields, and in particular to a kind of measuring method of human serum folic acid.

Background technique

Folic acid is important nutrient required for body, belongs to water soluble vitamin, participates in vivo acid, purine, phonetic The synthesis of pyridine.Earliest, folic acid is abnormal with prevention megaloblastic anemia, prevention embryo's nerve channel clinically mainly used for treating Shape.Currently, clinically common folic acid detection sample is mainly that serum folic acid detection and red blood cell acidum folicum detect 2 kinds.Serum leaf It is the index for reflecting recent folic acid intake that sour water is flat, can also clinically be used for the etiological diagnosis with cell anemia.It is red Cell folate can most reflect the long term storage situation of folic acid, and red blood cell acidum folicum is relatively low, then prompt folic acid long-term lacking.

Mainly there is the detection method of folate level in measurement human body: microbial method, chemiluminescence assay, isotope radiation Immunization, gas chromatography-mass spectrography, polymerase chain-restriction fragment length polymorphism measuring method, Ion capture and newest The mass spectrography for the folic acid derivatives being suggested, wherein chemiluminescence assay is a kind of clinically the most widely used side Method.The characteristics of high performance liquid chromatography is that measurement result is accurate, easy to operate, time saving and energy saving, analysis speed is fast, separating effect It is good, it is the method for the measurement folic acid having developed rapidly in recent years, analysis operating method is similar with drugs analysis used, is suitble to inspection Sterling is surveyed, but analysis cost is high, liquid chromatograph price and daily maintenance expense are expensive.Chemoluminescence method uses competitive binding receptor The basic principle of measurement, using folate binding protein as the basis of immune response.This method has the characteristics that quick, simplicity, should Method is easy to automate, result is repeated preferably, serum and red blood cell acidum folicum measurement can be used for simultaneously, to serum folic acid Measurement is not necessarily to pre-processing, and the measurement for red blood cell acidum folicum needs before measurement to handle whole blood sample, makes red thin Cellular lysis, while more glutamic acid folic acid in red blood cell are changed into single glutamic acid form.But need expensive precision instrument And reagent, sample handling processes are complex；In addition, enzyme-linked immunization testing result and actual numerical value are quite different.And micro- life Object Phacolysin detects biologically active folic acid quickly, this is that microbial method is different from the maximum feature of other methods and excellent Gesture, but there is also some defects for traditional microbiological method: and background is higher when such as folic acid is oxidized easily, detects.

Summary of the invention

The purpose of the present invention is to provide a kind of measuring methods of human serum folic acid, improve the accuracy of folic acid detection, Testing cost is reduced simultaneously.

To achieve the above object, technical solution of the present invention provides a kind of measuring method of human serum folic acid, including such as Lower step:

(1) preparation of experimental water: being added 4g active carbon powder in 1L deionized water, stirs 30s, and G4 sand core funnel filters Active carbon powder is removed, experimental water is obtained；

(2) folic acid titer is prepared: 200mg folic acid standard items are weighed, with 0.01mol/L ammonia solvent and are settled to 1L, The above-mentioned solution of 1mL is drawn, then with 0.01mol/L ammonia solvent and is settled to 1L, then draw 1mL, with 0.01mol/L ammonia solvent And it is settled to 25mL；It is returned to zero with 0.01mol/L ammonium hydroxide, cuvette optical path 1cm, colorimetric wavelength 256nm, according to ultraviolet absorptivity Value, calculates the folic acid concentration of the solution, then according to practical measurement concentration, is distinguished with 0.5% sodium ascorbate solution dilute It is interpreted as 0.0,0.1,0.2,0.3,0.4,0.5 and 0.6ug/L totally 7 kinds of standard solution；

(3) Lactobacillus rhamnosus growth medium prepare: weigh 4.7g folic acid culture medium dry powder, sodium ascorbate 0.05g, Chloramphenicol 0.02g, manganese sulfate 0.01g are put into 200mL beaker, add folic acid standard solution (the concentration 200ug/ that 100uL is to be calibrated L) and 100mL experimental water, it is heated to boiling after mixing, then high pressure sterilization, 121 DEG C of 20min obtain growth medium, cold But it is dispensed afterwards by 10mL, -20 DEG C freeze, the preparation for strain；

(4) preparation of Lactobacillus rhamnosus: Lactobacillus rhamnosus is inoculated in growth medium, training in 37 DEG C of incubators It supports for 24 hours, 2000r/min is centrifuged 2-3min, discards supernatant liquid, and growth medium is added, is centrifuged 2-3min after mixing, discards supernatant Growth medium is added in liquid, and for 24 hours, logarithmic growth phase bacterium solution and 80% glycerol are mixed in 1:1 ratio for culture in 37 DEG C of incubators It closes, 2mL cryovial is sub-packed in after mixing, -20 DEG C freeze；

(5) detection culture medium is prepared: being weighed 7.05g folic acid culture medium dry powder, chloramphenicol 0.003g, manganese sulfate 0.015g and is put Enter in 200mL beaker, 100mL experimental water is added, is heated to boiling, 75mg sodium ascorbate, 500uL sandlwood is added after cooling Sugared lactobacillus solution mixes；

(6) serum folic acid detect: by serum with 0.5% sodium ascorbate dilution 20 times in tissue culture plate, respectively plus Enter folic acid standard solution, blank well adds concentration to be the standard solution 100uL of 0.0ug/L, and sample well adds the serum to be checked after dilution The sodium azide solution that 5uL concentration is 3% is added in 100uL in blank well, and 200uL detection culture is added in Xiang Suoyou detection hole Base, sealed membrane blocks tissue culture plate, after tissue culture plate is set 37 DEG C of incubation 36h, mixes, tears sealed membrane, stands 5min, In microplate reader 590nm turbidimetric assay, suitable standard curve fit mode is selected to calculate the folic acid concentration of serum to be checked.

The present invention has the utility model has the advantages that it is dry to can achieve elimination using the organic impurities in activated carbon adsorption water by the present invention The purpose disturbed；The unstable chemcial property of folic acid, is oxidized easily, and ascorbic acid conduct is added in culture medium growing and detecting Antioxidant reduces the oxygen in hemolysate as far as possible, to increase the content of deoxyhemoglobin, reaches the work of protection folic acid With；Tissue culture plate is closed with sealed membrane when detection, facilitates the growth of Lactobacillus rhamnosus, can also prevent moisture evaporation and leaf The oxidation of acid；Simple and easy to do, cheap and high sensitivity of the invention, is more suitable for the folic acid screening needs of large-scale crowd.

Detailed description of the invention

Fig. 1 is the examination criteria curve of 1 folic acid of the embodiment of the present invention.

Specific embodiment

The technical scheme in the embodiments of the invention will be clearly and completely described below.