CN118329579A - Reagent for detecting trichomonas by adopting gram and Papanicolaou staining method - Google Patents
Reagent for detecting trichomonas by adopting gram and Papanicolaou staining method Download PDFInfo
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 71
- 241000224526 Trichomonas Species 0.000 title claims abstract description 60
- 238000007447 staining method Methods 0.000 title claims abstract description 32
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 101
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000007788 liquid Substances 0.000 claims abstract description 41
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 38
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 33
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000003223 protective agent Substances 0.000 claims abstract description 25
- 150000002191 fatty alcohols Chemical class 0.000 claims abstract description 17
- HGXBRUKMWQGOIE-AFHBHXEDSA-N (+)-pinoresinol Chemical compound C1=C(O)C(OC)=CC([C@@H]2[C@@H]3[C@@H]([C@H](OC3)C=3C=C(OC)C(O)=CC=3)CO2)=C1 HGXBRUKMWQGOIE-AFHBHXEDSA-N 0.000 claims abstract description 15
- 235000007221 pinoresinol Nutrition 0.000 claims abstract description 15
- OHOPKHNWLCMLSW-UHFFFAOYSA-N pinoresinol Natural products C1=C(O)C(OC)=CC(C2C3C(C(OC3)C=3C=C(CO)C(O)=CC=3)CO2)=C1 OHOPKHNWLCMLSW-UHFFFAOYSA-N 0.000 claims abstract description 15
- BURBOJZOZGMMQF-UHFFFAOYSA-N xanthoxylol Natural products C1=C(O)C(OC)=CC=C1C1C(COC2C=3C=C4OCOC4=CC=3)C2CO1 BURBOJZOZGMMQF-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229930040373 Paraformaldehyde Natural products 0.000 claims abstract description 13
- 229920002866 paraformaldehyde Polymers 0.000 claims abstract description 13
- 230000007935 neutral effect Effects 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 58
- 235000019441 ethanol Nutrition 0.000 claims description 31
- 238000004043 dyeing Methods 0.000 claims description 26
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 15
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 14
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 14
- 229910052740 iodine Inorganic materials 0.000 claims description 14
- 239000011630 iodine Substances 0.000 claims description 14
- 239000013078 crystal Substances 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 13
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000003794 Gram staining Methods 0.000 claims description 11
- 238000000605 extraction Methods 0.000 claims description 11
- 239000003761 preservation solution Substances 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 11
- 239000012192 staining solution Substances 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- VBIXEXWLHSRNKB-UHFFFAOYSA-N ammonium oxalate Chemical compound [NH4+].[NH4+].[O-]C(=O)C([O-])=O VBIXEXWLHSRNKB-UHFFFAOYSA-N 0.000 claims description 8
- 239000008213 purified water Substances 0.000 claims description 8
- JUQPZRLQQYSMEQ-UHFFFAOYSA-N CI Basic red 9 Chemical compound [Cl-].C1=CC(N)=CC=C1C(C=1C=CC(N)=CC=1)=C1C=CC(=[NH2+])C=C1 JUQPZRLQQYSMEQ-UHFFFAOYSA-N 0.000 claims description 7
- 239000008346 aqueous phase Substances 0.000 claims description 7
- 229940052223 basic fuchsin Drugs 0.000 claims description 7
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 6
- 229960000583 acetic acid Drugs 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 6
- 239000012898 sample dilution Substances 0.000 claims description 6
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 4
- 238000004042 decolorization Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 3
- 229920001612 Hydroxyethyl starch Polymers 0.000 claims description 3
- 229940103272 aluminum potassium sulfate Drugs 0.000 claims description 3
- 229960001506 brilliant green Drugs 0.000 claims description 3
- HXCILVUBKWANLN-UHFFFAOYSA-N brilliant green cation Chemical compound C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 HXCILVUBKWANLN-UHFFFAOYSA-N 0.000 claims description 3
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 229940050526 hydroxyethylstarch Drugs 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 claims description 3
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 claims description 3
- 235000010265 sodium sulphite Nutrition 0.000 claims description 3
- MPVDXIMFBOLMNW-ISLYRVAYSA-N 7-hydroxy-8-[(E)-phenyldiazenyl]naphthalene-1,3-disulfonic acid Chemical compound OC1=CC=C2C=C(S(O)(=O)=O)C=C(S(O)(=O)=O)C2=C1\N=N\C1=CC=CC=C1 MPVDXIMFBOLMNW-ISLYRVAYSA-N 0.000 claims description 2
- SEACYXSIPDVVMV-UHFFFAOYSA-L eosin Y Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 SEACYXSIPDVVMV-UHFFFAOYSA-L 0.000 claims description 2
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 claims description 2
- JLKDVMWYMMLWTI-UHFFFAOYSA-M potassium iodate Chemical compound [K+].[O-]I(=O)=O JLKDVMWYMMLWTI-UHFFFAOYSA-M 0.000 claims description 2
- 239000001230 potassium iodate Substances 0.000 claims description 2
- 235000006666 potassium iodate Nutrition 0.000 claims description 2
- 229940093930 potassium iodate Drugs 0.000 claims description 2
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 claims description 2
- 235000002639 sodium chloride Nutrition 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000009928 pasteurization Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 29
- 210000003495 flagella Anatomy 0.000 abstract description 17
- 238000010186 staining Methods 0.000 abstract description 16
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 22
- 230000000052 comparative effect Effects 0.000 description 13
- 241000224527 Trichomonas vaginalis Species 0.000 description 10
- 238000012360 testing method Methods 0.000 description 8
- 239000000975 dye Substances 0.000 description 7
- 235000011187 glycerol Nutrition 0.000 description 6
- 238000005457 optimization Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
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- 238000007789 sealing Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- QCDWFXQBSFUVSP-UHFFFAOYSA-N 2-phenoxyethanol Chemical compound OCCOC1=CC=CC=C1 QCDWFXQBSFUVSP-UHFFFAOYSA-N 0.000 description 1
- RSWGJHLUYNHPMX-UHFFFAOYSA-N Abietic-Saeure Natural products C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C(O)=O RSWGJHLUYNHPMX-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- KHPCPRHQVVSZAH-HUOMCSJISA-N Rosin Natural products O(C/C=C/c1ccccc1)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KHPCPRHQVVSZAH-HUOMCSJISA-N 0.000 description 1
- 208000005448 Trichomonas Infections Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- ZXJXZNDDNMQXFV-UHFFFAOYSA-M crystal violet Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1[C+](C=1C=CC(=CC=1)N(C)C)C1=CC=C(N(C)C)C=C1 ZXJXZNDDNMQXFV-UHFFFAOYSA-M 0.000 description 1
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- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 229960001235 gentian violet Drugs 0.000 description 1
- 239000000118 hair dye Substances 0.000 description 1
- 238000012835 hanging drop method Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
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- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- NALMPLUMOWIVJC-UHFFFAOYSA-N n,n,4-trimethylbenzeneamine oxide Chemical compound CC1=CC=C([N+](C)(C)[O-])C=C1 NALMPLUMOWIVJC-UHFFFAOYSA-N 0.000 description 1
- 229960005323 phenoxyethanol Drugs 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000009933 reproductive health Effects 0.000 description 1
- 210000005000 reproductive tract Anatomy 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 239000011697 sodium iodate Substances 0.000 description 1
- 235000015281 sodium iodate Nutrition 0.000 description 1
- 229940032753 sodium iodate Drugs 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- KHPCPRHQVVSZAH-UHFFFAOYSA-N trans-cinnamyl beta-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OCC=CC1=CC=CC=C1 KHPCPRHQVVSZAH-UHFFFAOYSA-N 0.000 description 1
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- 210000001215 vagina Anatomy 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a reagent for detecting trichomonas by adopting a gram and Pasteur staining method, which comprises a reagent liquid containing high-concentration ethanol or/and methanol or/and acetone or/and a sample treatment reagent liquid of water phase, wherein in order to maintain the structure of trichomonas, especially to protect the integrity of trichomonas flagella after staining, a protective agent A consisting of a mixed liquid of any two or more of chloroform, fatty alcohol, pinoresinol and diethyl ether is added into the reagent liquid, and a protective agent B consisting of a mixed liquid of any one or two of paraformaldehyde and neutral formaldehyde is also added into the sample treatment reagent liquid of water phase. The invention improves the reagent in the process of staining or sample treatment of the trichomonas, maintains the morphology of the trichomonas while ensuring the staining effect, and protects the trichomonas flagella from being damaged, thereby improving the sensitivity of detection and widening the clinical application range of the technology.
Description
Technical Field
The invention relates to the technical field of clinical detection of trichomonas, in particular to a detection reagent for detecting trichomonas by adopting a gram and Papanicolaou staining method.
Background
Trichomonas vaginalis parasitized in the vagina and urinary tract of a human body, is a protozoa microorganism with the length of 10-30 um and the width of 10-20 mu m, is colorless and transparent in living body, has refractive property, changeable body forms and strong mobility. After fixation dyeing, the hair dye is in pear shape or water drop shape, the front end is provided with a bubble-shaped nucleus, and the upper edge of the nucleus is provided with 5 matrixes which are arranged into a ring shape, so that 5 flagella are emitted: 4 anterior flagellums, 1 posterior flagellum. 1 axle column, which is thin and transparent, penetrates through the insect body and extends out of the body from the rear end. At the anterior 1/2 of the lateral aspect of the body, there is a surge membrane, the outer edge of which is attached to the posteriorly extending posterior flagellum. The insect body moves forward by means of the swinging of the flagella and makes rotary motion by the fluctuation of the fluctuation film.
Trichomonas vaginalis is a female urological and genital tract infectious disease caused by trichomonas vaginalis, and is also the most common non-viral transmission infection (STI) worldwide, leading to many bad reproductive health outcomes. In addition to the conventional hanging drop method, the clinical method for detecting trichomonas is also commonly used as a smear staining method, namely, taking vaginal side wall secretion, and using a gram or pasteurized smear to observe the trichomonas morphology in the secretion under a low power microscope. However, the problems of trichomonas breakage, flagella dissolution or falling off and the like are extremely easy to cause trichomonas omission in the treatment and dyeing processes of the sample by the two conventional dyeing methods. Therefore, the development of a reagent which can well maintain the trichomonas structure and improve the detection rate of trichomonas by the dyeing method is particularly important while ensuring the dyeing effect.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a reagent for detecting trichomonas by adopting gram and Papanicolaou staining methods. The reagent for detecting the trichomonas by adopting the gram and the Papanicolaou staining method can well maintain the structure of the trichomonas while ensuring the staining effect, and particularly protect the integrity of trichomonas flagella, thereby improving the detection rate of the gram and the Papanicolaou staining method to the trichomonas; in addition, the clinical application range of the technology is widened.
The invention is realized by the following technical scheme:
A reagent for detecting trichomonas by adopting a gram and Papanicolaou staining method comprises a reagent liquid containing more than 95% of high-concentration ethanol or/and methanol or/and acetone or/and a sample treatment reagent liquid of water phase, and a protective agent A consisting of a mixed solution of any two, three or four of chloroform, fatty alcohol, pinoresinol and diethyl ether is added into the reagent liquid of the high-concentration ethanol or/and methanol or/and acetone or/and the sample treatment reagent liquid of water phase in order to maintain the structure of trichomonas, especially to protect the integrity of trichomonas flagella after staining.
As optimization, a protective agent B consisting of paraformaldehyde or/and neutral formaldehyde or a mixture of two substances can be added into the sample treatment reagent liquid of the water phase.
As optimization, the concentration of the chloroform, the fatty alcohol and the pinoresinol in the reagent liquid is 5% -35%, and the concentration of the diethyl ether in the reagent liquid is 5% -50%; the concentration of the paraformaldehyde in the aqueous phase sample treatment reagent solution is 0.02% -0.4%, and the concentration of the neutral formaldehyde in the aqueous phase sample treatment reagent solution is 0.025% -5%.
As an optimization, the sample treatment reagent liquid of the water phase is a cell preservation liquid, a cell extraction liquid, a sample dilution liquid and a fixing liquid with a solvent being water.
Preferably, the fatty alcohol is ethylene glycol, n-butanol, tert-butanol or glycerol.
As an optimization, the gram staining method adopts a three-step method, specifically:
S1: the sample smear is primarily dyed by crystal violet dye liquor for 10s and then washed;
s2: mordant dyeing the sample smear obtained in the step S1 with iodine solution for 10S, and washing with water;
S3: re-dyeing the sample smear obtained in the step S2 with a decolorizing re-dyeing liquid for 2S, washing with water, airing and performing microscopic examination;
The preparation method of the crystal violet dye liquor comprises the steps of dissolving crystal violet in a mixture of ethanol and n-butanol, and stirring at room temperature to obtain liquor A; dissolving ammonium oxalate in purified water, and stirring until the ammonium oxalate is completely dissolved to obtain solution B; mixing the AB solution, standing, and filtering for 2 times; the preparation method of the iodine solution comprises the steps of dissolving potassium iodide in water, then adding iodine simple substance, mixing and stirring until iodine is completely dissolved, and storing in a brown bottle; the preparation method of the decolorizing double-dyeing liquid comprises the following steps: dissolving basic fuchsin in absolute ethyl alcohol, and stirring at room temperature to completely dissolve the basic fuchsin; then adding a protective agent A and acetone, which are composed of mixed liquid of any one or more of chloroform, fatty alcohol, pinoresinol and diethyl ether, and uniformly mixing.
As an optimization, the gram staining method adopts a four-step method, and the main reagents and components used comprise:
sample dilution: the main component is sodium chloride and water;
Crystal violet: the main components are crystal violet, ammonium oxalate, ethanol and water;
Iodine solution: the main components are iodine, potassium iodide and potassium iodate, and water:
decolorization liquid: the main components are acetone and ethanol;
And (3) a double-dyeing solution: the main components are safranine, basic fuchsin and ethanol, and water.
Wherein, a protective agent A consisting of any one or more than one of chloroform, fatty alcohol, pinoresinol and diethyl ether or a protective agent B consisting of any one or two of paraformaldehyde and/or neutral formaldehyde is added into the sample diluent.
As an optimization, when the papanicolaou staining method is adopted, the main reagents and components used include:
cell preservation solution: the main components are tris (hydroxymethyl) aminomethane, sodium chloride, ethanol, sodium sulfite and water;
Cell extract: the main components are disodium ethylenediamine tetraacetate, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, hydroxyethyl starch, dithiothreitol and water;
Hematoxylin: the main components are hematoxylin, aluminum potassium sulfate, glacial acetic acid and water;
EA/OG staining solution: the main components are eosin Y, brilliant green SF, orange G, phosphotungstic acid, acetic acid, ethanol, methanol and water;
an ethanol solution for immobilization.
Wherein, the protective agent A consisting of any one or a mixture of any two, three or four of chloroform, fatty alcohol, pinoresinol and diethyl ether is added into the cell preservation solution, the cell extraction solution, the EA/OG staining solution and the ethanol solution for fixation; and a protective agent B consisting of paraformaldehyde or/and neutral formaldehyde or a mixture of two substances can be added into the cell preservation solution and the cell extraction solution of the aqueous phase sample treatment reagent.
The dyeing process is the same as the conventional Papanicolaou dyeing method, and comprises the following operation steps: cell preservation, cell extraction, cell fixation, buffer environment, hematoxylin staining, buffer environment, EA/OG staining solution, dehydration, transparency and sealing and preservation.
The beneficial effects of the invention are as follows:
The reagent for detecting trichomonas by adopting the gram and Papanicolaou staining method provided by the invention has the advantages that the reagent for detecting trichomonas by adopting the gram and Papanicolaou staining method is improved by containing more than 95% of ethanol or/and methanol or/and acetone or/and sample treatment in the staining method, the complete morphological structure of trichomonas is maintained while the staining effect is ensured, so that the flagella are clearly visible, the detection rate of the gram staining method and the Papanicolaou staining method on the trichomonas is improved, and the clinical application range of the technology is widened.
Drawings
A reagent for detecting trichomonas by the gram and Papanicolaou staining method is further described below with reference to the accompanying drawings:
FIG. 1 is a 100-fold field of view of an oil mirror (arrow pointing to Trichomonas vaginalis) after staining with example 1 of the present invention;
FIG. 2 is a 100-fold field of view of the oil mirror after staining with comparative example 1 (arrow pointing to Trichomonas vaginalis);
FIG. 3 is a 100-fold field of view of an oil mirror (arrow pointing to trichomonas vaginalis) after staining with example 2 of the present invention using a conventional gram staining method;
FIG. 4 is a 100-fold field of view of the oil mirror (arrow pointing to trichomonas vaginalis) after staining with comparative example 2 using a conventional gram staining method;
FIG. 5 is a 100-fold field of view of the scope (arrow pointing to Trichomonas vaginalis) after staining with example 3 of the present invention using the Papanicolaou staining method;
Fig. 6 is a 100-fold field of view (arrow pointing to trichomonas vaginalis) using the papanicolaou staining method after staining with comparative example 3.
Detailed Description
The application is described in further detail below with reference to the drawings and examples. It is specifically noted that the following examples are only for illustrating the present application, but do not limit the scope of the present application. Likewise, the following examples are only some, but not all, of the examples of the present application, and all other examples, which a person of ordinary skill in the art would obtain without making any inventive effort, are within the scope of the present application.
In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art.
Example 1: detection of trichomonas in a sample using the gram stain three-step method of the present invention and the detection reagent of the present invention
1. Test solution preparation
1. Crystal violet dye liquor: dissolving 1g of crystal violet in a mixture of 185ml of ethanol and 75ml of n-butanol, and stirring at room temperature for 6 hours to obtain solution A; dissolving 4g of ammonium oxalate in 740ml of purified water, and stirring until the ammonium oxalate is completely dissolved to obtain solution B; mixing the AB solution, standing for 48h, and filtering for 2 times for standby;
2. iodine solution: dissolving 6.7g of potassium iodide in 500ml of water, then adding 3.3g of iodine simple substance, mixing and stirring for 3 hours until the iodine is completely dissolved, fixing the volume to 1000ml by using water, and storing in a brown bottle for standby;
3. decolorizing double-dyeing liquid: 3.57g of basic fuchsin is dissolved in 500ml of absolute ethyl alcohol and stirred for 1h at room temperature to be completely dissolved; then 200ml chloroform and 300ml acetone were added and mixed well for use.
In the same way, in actual preparation, the protective agent A in the decolorized complex dye solution can be any one or a mixture of any two, three or four of chloroform, fatty alcohol, pinoresinol and diethyl ether, and the effect is consistent.
2. Dyeing method
1. The sample smear is primarily dyed for 10s by crystal violet dye liquor and washed;
2. Mordant dyeing the sample smear obtained in the step 1 for 10s by using iodine solution, and washing the sample smear by using water;
3. And (3) counterstaining the sample smear obtained in the step (2) with a decolorizing counterstain solution for 2s, washing with water, and airing for microscopic examination.
3. Detection result
The smear was observed with a 100X oil microscope, and the result is shown in FIG. 1, wherein the trichomonas structure is clear and the flagella are visible.
Comparative example 1: in contrast, the same sample was stained as in example 1, wherein 200ml of chloroform was not added to the decolorized complex stain solution, and the other steps were the same as in example 1. The result after staining is shown in FIG. 2, no visible flagella is found, and the result is easily confused with white blood cells and pus cells.
The comparison result proves that after chloroform is added into the decolorized counterstain liquid, the structure of trichomonas is effectively protected, and the detection sensitivity is improved.
Example 2: detection of trichomonas in a sample using conventional gram staining methods and detection reagents of the present invention
1. Test solution preparation
1. Sample dilution: 9g of sodium chloride is dissolved in 1000ml of 0.2% paraformaldehyde solution for standby;
2. A commercial conventional gram stain kit was used.
In the same way, in actual preparation, the protective agent used in the sample diluent can be any one or two substances of protective agent B paraformaldehyde or/and neutral formaldehyde, and the protective agent A can be any one or two, three or four substances of chloroform, fatty alcohol, rosin alcohol and diethyl ether.
2. Dyeing method
1. Placing the swab with the sample into a sample diluent bottle, absorbing a sample smear after full elution, or dripping the sample diluent on a glass slide after direct smear to cover a sample area;
2. The smear to be stained is gram four-step stained according to the commodity instructions: primary dyeing of gentian violet dye liquor for 15s, and washing; mordant for 15s, washing with water; decolorizing the decolorized solution until no color flows down, and washing with water; and (5) counterstaining the yellow sand for 10s, washing with water, airing, and microscopic examination. It should be noted that the decoloring time should not exceed 15s.
3. Detection result
The smear was observed with a 100X oil microscope, and the result is shown in FIG. 3, wherein the trichomonas structure is clear and the flagella are visible.
Comparative example 2: in contrast, the same sample was stained as in example 2, wherein no paraformaldehyde was added to the sample dilution, and the other samples were stained as in example 2. The result after staining is shown in FIG. 4, no visible flagella is found, and the result is easily confused with white blood cells and pus cells.
It can be seen that the detection reagent of the present invention can be used alone when the smear is fixed, and can be more conveniently adapted to the conventional gram staining reagent solution described above without modification.
Example 3: detection of trichomonas in a sample using Papanicolaou staining method and detection reagent of the present invention
1. Test solution preparation
1. Cell preservation solution: 5g of tris (hydroxymethyl) aminomethane, 2g of sodium chloride and 1g of sodium sulfite were dissolved in 700ml of purified water, and 300ml of absolute ethyl alcohol and ethylene glycol (ethylene glycol: absolute ethyl alcohol=1:5) were added to obtain a reagent liquid a.
2. Cell extract: 2g of disodium ethylenediamine tetraacetate, 0.6g of disodium hydrogen phosphate, 1.5g of potassium dihydrogen phosphate, 7g of sodium chloride, 0.2g of potassium chloride, 5g of hydroxyethyl starch and 0.1g of dithiothreitol are dissolved in 899ml of purified water, and 1ml of phenoxyethanol and 100ml of glycerin are added to obtain a reagent solution B.
3. Hematoxylin dye liquor: 1.6g of hematoxylin is dissolved in 189ml of absolute ethyl alcohol, 14g of aluminum potassium sulfate is dissolved in 800ml of purified water, the two are uniformly mixed, and 1.6g of sodium iodate, 0.16g of disodium edetate, 15ml of glacial acetic acid and 38ml of glycerin are added to the mixture to obtain a reagent solution C.
4. EA/OG staining solution: eosin Y4G, brilliant green SF 0.75G and orange G0.5G were dissolved in 60ml of purified water to obtain ①, phosphotungstic acid 2G was dissolved in 20ml of purified water to obtain ②, methanol 200ml, absolute ethanol 600ml and pinoresinol 65ml were uniformly mixed to obtain ③, ①、② was poured into ③ and uniformly mixed, and acetic acid 30ml and glycerin 25ml were added to obtain reagent liquid D.
5. Fixing solution: and (3) mixing 950ml of 95% ethanol and 50ml of diethyl ether uniformly to obtain a reagent solution E.
Similarly, in the actual preparation, the protective agent A used in the cell preservation solution, the cell extraction solution, the EA/OG staining solution and the ethanol solution for fixation may be any one or a mixture of any two, three or four of chloroform, fatty alcohol, pinoresinol and diethyl ether, and the fatty alcohol may be ethylene glycol, n-butanol, t-butanol or glycerol. In addition, the cell preservation solution and the cell extraction solution of the aqueous phase treatment reagent can also adopt a protective agent B, namely, paraformaldehyde or/and neutral formaldehyde or a mixed solution of any two substances, and the use effects of the protective agent B and the mixed solution are consistent.
2. Dyeing method
1. Placing the sampled sample brush/swab into a reagent solution A, and transferring to a laboratory;
2. Sucking 4ml of the reagent solution B into a centrifuge tube, transferring 8ml of the reagent solution A stored with the sample into the centrifuge tube slowly, balancing, and centrifuging for 5min by 800G to enrich the sample;
3. Transferring the enriched sample to a sedimentation bin on a glass slide, and fixing for 10min by using a reagent solution E;
4. Removing liquid in the sedimentation bin, adding Tris buffer for rinsing for 2 times, discarding, then dyeing for 70s by using reagent liquid C, discarding, continuing rinsing with the Tris buffer for 2 times, and discarding;
5. washing with 95% ethanol solution for 2 times, discarding, then dyeing with reagent solution D for 2min, discarding, washing with 95% ethanol for 3 times, discarding;
6. And (3) washing the slide with absolute ethyl alcohol, clearing the slide with xylene/novel transparent agent for 3min, sealing the slide with neutral resin, and airing and microscopic examination.
3. Detection result
The smear was observed with a 100X oil microscope, and the results are shown in FIG. 5, wherein the trichomonas structure is clear and the flagella are visible.
Comparative example 3: in contrast, the results of staining the same sample by the conventional papanicolaou staining method are shown in fig. 6, and the same sample is substantially free of visible flagella and easily confused with white blood cells and basal layer cells.
The comparison result proves that after the ethylene glycol, the glycerol, the pinoresinol and the diethyl ether are respectively added into the cell preservation solution, the cell extraction solution, the EA/OG staining solution and the ethanol solution for fixation, the trichomonas structure is effectively protected, and the detection sensitivity is improved.
Example 4: clinical comparative statistics
114 Clinical female vaginal secretions were collected, of which the clinically definite diagnosis was 26 total cases of trichomonas infection or trichomonas combined with other types of infection, the remaining 88 cases being non-trichomonas samples. The batch was numbered 114 samples, each sample being tested for control according to example 1, example 2 and example 3, respectively. The statistical results are shown in the following table:
Table 1 comparison of example 1 with clinical diagnostic results
Conclusion: by adopting the method of the embodiment 1 of the invention for detection, 1 case of trichomonas missed detection is detected, the sensitivity of trichomonas detection is 96.2%, and the specificity is 100%.
TABLE 2 comparison of example 2 with clinical diagnostic results
Conclusion: when the method of the embodiment 2 is adopted for detection, 2 cases of trichomonas missed detection are detected, the sensitivity of trichomonas detection is 92.3%, and the specificity is 100%.
TABLE 3 comparison of example 3 with clinical diagnostic results
Conclusion: when the method of the embodiment 3 is adopted for detection, 2 cases of trichomonas missed detection are detected, the sensitivity of trichomonas detection is 92.3%, and the specificity is 100%.
Table 4 comparison of comparative example 1 with clinical diagnostic results
Conclusion: when the test was carried out by the method of comparative example 1, the test was omitted for 12 cases of trichomonas, the sensitivity of the test was 53.8%, and the specificity was 100%.
Table 5 comparative example 2 and clinical diagnostic results comparison
Conclusion: when the test was carried out by the method of comparative example 2, the sensitivity of the test was 57.7% and the specificity was 100% in 11 cases of trichomonas omission.
TABLE 6 comparison of comparative example 3 with clinical diagnostic results
Conclusion: when the test was carried out by the method of comparative example 3, the test was omitted for 15 cases of trichomonas, the sensitivity of the test was 42.3%, and the specificity was 100%.
As can be seen from the experimental comparison data, the detection reagent for detecting trichomonas by adopting the gram and Papanicolaou staining method provided by the invention effectively improves the detection sensitivity of the gram staining and Papanicolaou staining methods of trichomonas vaginalis. In addition, when the existing commercial gram staining solution is used, the structure of the trichomonas can be protected by using the method provided by the invention when the sample is diluted and fixed, so that the clinical application range of the technology is further widened.
The above description shows the technical idea and features of the present invention. It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments or examples, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing implementations or examples should be regarded as illustrative rather than limiting. The scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any simple modification and equivalent variation of the above embodiments according to the technical substance of the present invention should fall within the scope of the present invention without departing from the technical principle of the present invention.
Claims (10)
1. A reagent for detecting trichomonas by adopting a gram and pasteurization method, comprising a reagent liquid of high-concentration ethanol or/and methanol or/and acetone or/and a sample treatment reagent liquid of water phase, which is characterized in that: the protective agent A consisting of a mixed solution of any one or any two, three or four of chloroform, fatty alcohol, pinoresinol and diethyl ether is added into the high-concentration ethanol or/and methanol or/and acetone reagent solution or/and water phase sample treatment reagent solution, wherein:
When the gram dyeing method is adopted, the protective agent A is added into a sample diluent or/and a decolorization solution or/and a decolorization double-dyeing solution;
When the Papanicolaou staining method is adopted, the protective agent A is added into the cell preservation solution, the cell extraction solution, the EA/OG staining solution and the ethanol solution for fixation.
2. A reagent for detecting trichomonas by the gram and pap staining method according to claim 1, wherein: the fatty alcohol is ethylene glycol, n-butanol, tert-butanol or glycerol.
3. A reagent for detecting trichomonas by the gram and pap staining method according to claim 2, wherein: the concentration of chloroform, fatty alcohol and pinoresinol in the reagent liquid is 5% -35%, and the concentration of diethyl ether in the reagent liquid is 5% -50%.
4. A reagent for detecting trichomonas by the gram and pap staining method according to any one of claims 1 to 3, wherein: and a protective agent B consisting of paraformaldehyde or/and neutral formaldehyde or a mixed solution of two substances can be added into the aqueous sample treatment reagent solution.
5. A reagent for detecting trichomonas by the gram and pap staining method according to claim 4, wherein: the aqueous sample treatment reagent liquid is a sample dilution liquid, a cell preservation liquid, a cell extraction liquid and a fixing liquid with a solvent being water.
6. A reagent for detecting trichomonas by the gram and pap staining method according to claim 5, wherein: the concentration of the paraformaldehyde in the aqueous phase sample treatment reagent solution is 0.02% -0.4%, and the concentration of the neutral formaldehyde in the aqueous phase sample treatment reagent solution is 0.025% -5%.
7. A reagent for detecting trichomonas by the gram and pap staining method according to claim 6, wherein: the gram staining method adopts a three-step method, and specifically comprises the following steps:
S1: the sample smear is primarily dyed by crystal violet dye liquor for 10s and then washed;
s2: mordant dyeing the sample smear obtained in the step S1 with iodine solution for 10S, and washing with water;
S3: re-dyeing the sample smear obtained in the step S2 with a decolorizing re-dyeing liquid for 2S, washing with water, airing and performing microscopic examination;
the preparation method of the crystal violet dye liquor comprises the steps of dissolving crystal violet in a mixture of ethanol and n-butanol, and stirring at room temperature to obtain liquor A; dissolving ammonium oxalate in purified water, and stirring until the ammonium oxalate is completely dissolved to obtain solution B; mixing the AB solution, standing, and filtering for 2 times;
The preparation method of the iodine solution comprises the steps of dissolving potassium iodide in water, then adding iodine simple substance, mixing and stirring until iodine is completely dissolved, and storing in a brown bottle;
the preparation method of the decolorizing double-dyeing liquid comprises the following steps: dissolving basic fuchsin in absolute ethyl alcohol, and stirring at room temperature to completely dissolve the basic fuchsin; and then adding the protective agent A and acetone which are composed of any one or more than one of chloroform, fatty alcohol, pinoresinol and diethyl ether, and uniformly mixing.
8. A reagent for detecting trichomonas by the gram and pap staining method according to claim 6, wherein: the gram staining method adopts a four-step method, and the main reagents and components used comprise:
sample dilution: the main component is sodium chloride and water;
Crystal violet: the main components are crystal violet, ammonium oxalate, ethanol and water;
Iodine solution: the main components are iodine, potassium iodide and potassium iodate, and water:
decolorization liquid: the main components are acetone and ethanol;
Counterstaining liquid: the main components are safranine, basic fuchsin and ethanol, water;
Wherein the protective agent A consisting of any one or a mixture of any two, three or four of chloroform, fatty alcohol, pinoresinol and diethyl ether or the protective agent B consisting of any one or two of paraformaldehyde and/or neutral formaldehyde is added into the sample diluent.
9. A reagent for detecting trichomonas by the gram and pap staining method according to claim 6, wherein: the main reagents and components of the Papanicolaou staining method comprise:
cell preservation solution: the main components are tris (hydroxymethyl) aminomethane, sodium chloride, ethanol, sodium sulfite and water;
Cell extract: the main components are disodium ethylenediamine tetraacetate, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, hydroxyethyl starch, dithiothreitol and water;
Hematoxylin: the main components are hematoxylin, aluminum potassium sulfate, glacial acetic acid and water;
EA/OG staining solution: the main components are eosin Y, brilliant green SF, orange G, phosphotungstic acid, acetic acid, ethanol, methanol and water;
An ethanol solution for immobilization;
Wherein the protective agent A consisting of any one or a mixture of any two, three or four of chloroform, fatty alcohol, pinoresinol and diethyl ether is added into the cell preservation solution, the cell extraction solution, the EA/OG staining solution and the ethanol solution for fixation; the protective agent B composed of any one or a mixture of two substances of paraformaldehyde and/or neutral formaldehyde can be added into the cell preservation solution and the cell extraction solution of the aqueous phase sample treatment reagent.
10. A reagent for detecting trichomonas by the gram and pap staining method according to claim 1, wherein: the reagent solution of the high-concentration ethanol or/and methanol or/and acetone is reagent solution containing more than 95% of the high-concentration ethanol or/and methanol or/and acetone.
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