CN101565728A - Nutritional digestive juice for chlamydia neutralization - Google Patents

Abstract

The invention relates to a Nutritional digestive juice for chlamydia neutralization, in particular to a prescription of a cell culture medium used when a urethritis patient needs the examination of the chlamydia cell culture after using a large amount of clinical antibiotics and a preparation method of the cell culture medium. The genital tract chlamydia trachomatis infection is one of the most common diseases which are sexually transmitted. Chlamydia trachomatis is also regarded as the important cause of the pelvic inflammatory disease and the sequelae (tubal infertility and ectopic pregnancy) thereof. The chlamydia cell culture method is the most sensitive and the most reliable method used for examining the chlamydia. The culture medium can detect out the positive result of the genital tract chlamydia after the large amount of clinical antibiotics is used, thereby having the function of the clinical diagnosis guidance and the clinical treatment and the important meaning of preventing the abuse of the antibacterial drugs.

Description

In the chlamydozoan and nutritional digestive juice

Technical field

The present invention relates in a kind of chlamydozoan and nutritional digestive juice; Particularly the urethritis patient behind clinical a large amount of use microbiotic when needing the chlamydozoan cell cultures to check, uses prescription of cell culture medium and preparation method thereof.Its prescription main component is: soil temperature-80, lecithin, sodium-chlor, Sodium phosphate dibasic, potassium primary phosphate, Repone K, sal epsom, glucose, calcium chloride, lactalbumin hydrolysate, calf serum, 1% glutamine, 2 mercapto ethanol, penicillin, Streptomycin sulphate, gentamicin, EDTA, 0.5% phenol red.The chlamydia trachomatis genital tract infection is one of the most common sexually transmitted disease.Chlamydia trachomatis also is considered to pelvic inflammatory disease and sequela thereof---the important cause of disease of the infertile and ectopic pregnancy of uterine tube.The pregnant woman suffers from the chlamydia trachomatis cervicitis, and fetus can infect chlamydia trachomatis conjunctivitis and pneumonia when birth canal is given a birth.The chlamydozoan cell culture method is to check responsive, the most reliable method of chlamydozoan.Especially to chronic patients and female patient, need after cultivating, just can make correct diagnosis; The patient who has treated is needed to confirm whether fully recover through cultivating one's ability.This substratum can detect the reproductive tract chlamydozoan positive findings behind a large amount of clinical use microbiotic, has the effect of guiding clinical diagnosis and treatment, and is also significant for preventing to abuse antibacterials.McCoy cell commonly used or Hela229 monolayer cell are cultivated, and can determine diagnosis by characteristic inclusion body in observation of cell pathology (enlargement, sex change come off) and the cell.Its susceptibility is about 80%～90%, and specificity is 100%.And can be used for drug sensitivity test, also can detect treatment back patient and whether have chlamydozoan alive, to judge curative effect.This method operation is cumbersome, time-consuming, and apparatus expensive is technical strong, clinical difficult popularization.Chlamydia trachomatis need be cooked the cell cultivation in the laboratory and separate, therefore, cell cultures " gold standard " that always detect for many years as chlamydozoan, cell cultures is all very high to equipment, technology, collection of specimens and the requirement of transporting etc., mast cell from urethra, uterine neck collection, syncyte and mucopurulent secretion thing should not be mixed with, too much impurity should not be contained.To gathering swab requirement is arranged also: should be cotton swab and should not be wooden swab, because wooden having wiped away may be suppressed the chlamydozoan growth.In the uterine neck sample have cytobrush can collect more from cell to improve the susceptibility of cultivating.Sample must be placed in the special transhipment training base, and is freezing, is embedded in the cell cultures dish in 24 hours.The state of McCoy cell also can influence net result during cultivation.When the McCoy cell transition is grown, is out of shape, breaks even fully during the lost cell form, the inclusion body in the observation of cell of then having no way of.Cell cultures in cost, equipment, technical elements all requires very highly, for cell cultures, cost and time are to influence the biggest obstacle that the chlamydozoan cell cultures is used and promoted as diagnostic method.A lot of researchists the improvement of cell culture processes enterprising many-sided exploration.

Background technology

The urogenital system disease that is caused by chlamydia trachomatis is one of pathogenic agent that more and more causes in recent years clinical attention.Especially a large amount of clinical with the very difficult inspection of the detection of the chlamydia trachomatis behind the microbiotic, owing to contain the abundant residues microbiotic in the sample, when recovering to cultivate chlamydia trachomatis, continue to be subjected to the effect of residues of antibiotics.Therefore, do not adopt the experiment of removing remaining microbiotic measure, should think serious mistake in design, the result who draws according to such experiment is insecure.Cell cultures in cost, equipment, technical elements all requires very highly, for cell cultures, cost and time are to influence the biggest obstacle that the chlamydozoan cell cultures is used and promoted as diagnostic method.At present, cell cultures is still diagnosis clothing former full infection reliable experiment method the most, traditionally cell cultures as " gold standard ".Determine that with the method for cell cultures chlamydial infection relates to the influence factor of 5 aspects: 1. gather as far as possible and contain more epithelial sample; 2. in transhipment training base, add microbiotic to prevent the hypertrophy of bacterium in the sample; 3. 4. use antimetabolite to suppress histiocytic duplicating; 5. use iodine, Giemsa, immunofluorescence dyeing to identify chlamydozoan in the cells infected.The object of the present invention is to provide in a kind of chlamydozoan and nutritional digestive juice, compare with other class reagent and to possess following characteristics: in the chlamydozoan and nutritional digestive juice is nutritious, preparation is simple, selectivity is strong and variable color is sensitive, compare with conventional reagents, the recall rate height, pollute for a short time, clinical application effect is satisfied. and detection method is simple, the time is short, accuracy is strong.Can prevent the hypertrophy of bacterium in the sample, duplicating of cultured tissue cell suppressed.

Summary of the invention

Main points of the present invention are to select suitable component, and rationally are mixed, through heat, dissolve, technology such as mixing, cooling, packing, high-temperature sterilization forms in a kind of chlamydozoan and nutritional digestive juice.

In the chlamydozoan that the present invention selects and nutrition digestion liquid formula following (gram, every liter of distilled water of ml/): soil temperature-800.3-0.5; Lecithin 0.5-0.8; Sodium-chlor 70.0-90.0; Sodium phosphate dibasic 0.5-0.7; Potassium primary phosphate 0.5-0.7; Repone K 3.0-5.0; Sal epsom 1.5-3.0 glucose 8.0-12.0; Calcium chloride 1.0-2.0; Lactalbumin hydrolysate 2.0-3.0; Calf serum 10.0-12.0ml; 1% glutamine 1.0-1.2ml; 2 mercapto ethanol 0.5-0.6; Penicillin, Streptomycin sulphate, gentamicin (10000U/ml) 10.0-15.0ml; EDTA 0.1-0.3; 0.5% phenol red 3.0-5.0ml; Distilled water adds to 1000ml; Transfer pH to 7.2-7.4.

Soil temperature-80, lecithin are neutral neutralizing agents, can be effectively with the absorption of the residues of antibiotics in sample neutralization, and neutralized reaction product does not have influence to culture.

Sodium-chlor, Sodium phosphate dibasic, potassium primary phosphate, Repone K, sal epsom, glucose, calcium chloride, be the requisite material of histiocytic body fluid equilibrium; Lactalbumin hydrolysate, calf serum, 1% glutamine, be the requisite material of histiocytic nutritive equilibrium; Penicillin, Streptomycin sulphate, gentamicin are to prevent to pollute requisite material in the cell cultivation process; 2 mercapto ethanol, EDTA play the digestion of tissue block to improve the output of cell in the cell cultivation process.Want before calf serum uses deactivation (56 ℃, 30min), to eliminate complement activity.The animal serum individual difference is big, detects so each batch serum all wants strict, carries out sterility test simultaneously.High-quality serum should be for faint yellow, and transparent, no haemolysis does not have precipitation, and color is dark slightly after the deactivation.Biochemistry detection total protein content 3.5 ~ 4.5g/100ml, sphaeroprotein is not higher than 2g/100ml, warranty test condition stable.After adding calf serum in the test, the pH of substratum may also can change, so also can add adjust pH behind the calf serum earlier.After nutrient solution prepares, should extract a little earlier and put into culturing bottle,, whether pollution be arranged to detect nutrient solution in 37 ℃ of built-in 24～48hr of incubator.Each dosing amount is advisable about with two weeks, and a dosing is not too many, prevents nutritive ingredient (being mainly glutamine) loss, causes experiment loaded down with trivial details or pollute.

The preparation method of product of the present invention is:

1), soil temperature-80, lecithin, sodium-chlor, Sodium phosphate dibasic, potassium primary phosphate, Repone K, sal epsom, glucose, EDTA are heated in proportion, dissolves;

2), with calcium chloride in proportion heating for dissolving in distilled water, be chilled to 54 ℃;

3), with dissolved 1) mixed solution slowly joins dissolved 2) in the solution, (noting precipitation) mixes, PH7.2-7.4 is transferred in the cooling back, adds phenol red;

4), dissolved 1% glutamine, 2 mercapto ethanol are added in the above-mentioned mixed solution in proportion, 115 ℃ of pressuresteam sterilizations, 20-30min is chilled to 50 ℃;

5), adding aseptic lactalbumin hydrolysate, calf serum, mixing microbiotic under aseptic condition mixes; Aseptic subpackaged, refrigerate standby.

The present invention has the following advantages: in (1) chlamydozoan and nutritional digestive juice, preparation is simple, and starting material are easy to get; (2) clinical application effect is satisfied, has solved the difficulty of bedside inoculation, pollutes little; (3) detection method is simple, the time is short, selectivity is strong, variable color is sensitive, recall rate is high; (4) can prevent the hypertrophy of bacterium in the sample, duplicating of cultured tissue cell suppressed.Can in and contain the abundant residues microbiotic in the sample, the patient that treated is needed to confirm whether fully recover, have guiding clinical diagnosis and therapeutic action through cultivating one's ability, also significant for preventing to abuse antibacterials.

Embodiment

The present invention is described in further detail below in conjunction with embodiment:

Be configured according to following table institute column data (gram, every liter of distilled water of ml/) and described step thereof:

Prescription one:

Soil temperature-800.3-0.5; Lecithin 0.5-0.8; Sodium-chlor 70.0-90.0; Sodium phosphate dibasic 0.5-0.7; Potassium primary phosphate 0.5-0.7; Repone K 3.0-5.0; Sal epsom 1.5-3.0 glucose 8.0-12.0; Calcium chloride 1.0-2.0; Lactalbumin hydrolysate 2.0-3.0; Calf serum 10.0-12.0ml; 1% glutamine 1.0-1.2ml; 2 mercapto ethanol 0.5-0.6; Penicillin, Streptomycin sulphate, gentamicin (10000U/ml) 10.0-15.0ml; EDTA 0.1-0.3; 0.5% phenol red 3.0-5.0ml; Distilled water adds to 1000ml; Transfer pH to 7.2-7.4.

Prescription two:

Soil temperature-800.3-0.4; Lecithin 0.5-0.7; Sodium-chlor 70.0-80.0; Sodium phosphate dibasic 0.6-0.7; Potassium primary phosphate 0.6-0.7; Repone K 4.0-5.0; Sal epsom 2.5-3.0 glucose 9.0-12.0; Calcium chloride 1.5-2.0; Lactalbumin hydrolysate 2.5-3.0; Calf serum 11.0-12.0ml; 1% glutamine 1.1-1.2ml; 2 mercapto ethanol 0.5-0.6; Penicillin, Streptomycin sulphate, gentamicin (10000U/ml) 12.0-15.0ml; EDTA 0.2-0.3; 0.5% phenol red 4.0-5.0ml; Distilled water adds to 1000ml; Transfer pH to 7.2-7.4.

Prescription three:

Soil temperature-800.3-0.4; Lecithin 0.6-0.8; Sodium-chlor 80.0-90.0; Sodium phosphate dibasic 0.5-0.6; Potassium primary phosphate 0.5-0.6; Repone K 3.0-4.0; Sal epsom 1.5-2.0 glucose 8.0-10.0; Calcium chloride 1.0-1.5; Lactalbumin hydrolysate 2.0-2.5; Calf serum 10.0-11.0ml; 1% glutamine 1.0-1.1ml; 2 mercapto ethanol 0.5-0.6; Penicillin, Streptomycin sulphate, gentamicin (10000U/ml) 10.0-13.0ml; EDTA 0.1-0.2; 0.5% phenol red 3.0-4.0ml; Distilled water adds to 1000ml; Transfer pH to 7.2-7.4.

Claims (2)

1, the present invention relates in a kind of chlamydozoan and nutritional digestive juice; Particularly the urethritis patient behind clinical a large amount of use microbiotic when needing the chlamydozoan cell cultures to check, uses prescription of cell culture medium and preparation method thereof, it is characterized in that having following prescription (gram, every liter of distilled water of ml/): soil temperature-800.3-0.5; Lecithin 0.5-0.8; Sodium-chlor 70.0-90.0; Sodium phosphate dibasic 0.5-0.7; Potassium primary phosphate 0.5-0.7; Repone K 3.0-5.0; Sal epsom 1.5-3.0 glucose 8.0-12.0; Calcium chloride 1.0-2.0; Lactalbumin hydrolysate 2.0-3.0; Calf serum 10.0-12.0ml; 1% glutamine 1.0-1.2ml; 2 mercapto ethanol 0.5-0.6; Penicillin, Streptomycin sulphate, gentamicin (10000U/ml) 10.0-15.0ml; EDTA 0.1-0.3; 0.5% phenol red 3.0-5.0ml; Distilled water adds to 1000ml; Transfer pH to 7.2-7.4.

2, in the described chlamydozoan of a kind of claim 1 and the preparation method of nutritional digestive juice, it is characterized in that having following step:

1), soil temperature-80, lecithin, sodium-chlor, Sodium phosphate dibasic, potassium primary phosphate, Repone K, sal epsom, glucose, EDTA are heated in proportion, dissolves;

2), with calcium chloride in proportion heating for dissolving in distilled water, be chilled to 54 ℃;

3), with dissolved 1) mixed solution slowly joins dissolved 2) in the solution, (noting precipitation) mixes, PH7.2-7.4 is transferred in the cooling back, adds phenol red;

4), dissolved 1% glutamine, 2 mercapto ethanol are added in the above-mentioned mixed solution in proportion, 115 ℃ of pressuresteam sterilizations, 20-30min is chilled to 50 ℃;

5), adding aseptic lactalbumin hydrolysate, calf serum, mixing microbiotic under aseptic condition mixes; Aseptic subpackaged, refrigerate standby.