Keywords: genital warts laboratory tests
, Acetic acid white test
Coated with 3-5% acetic acid wart for 2-5 minutes, the lesion sites whiten little bulge, anal lesions may take 15 minutes. The principle of this test is the result of protein and acid coagulation whiten HPV infected cells produce keratin uninfected normal epithelial cells to produce a different, only the former can be acetate decolorization. Acetic acid white test to detect HPV is highly sensitive than conventional detection observed histological changes better. But occasionally false-positive epithelial thickening or abrasions trauma cases, false positive turn white signs seem ill-defined and irregular. U.S. CDC suggest that acetic acid white test is not a specific test, and false positives are more common.
Second, the immunization histological examination
Top peroxidase anti-peroxidase method (i.e., PAP), the viral proteins within the display condyloma, to demonstrate that the viral antigen in the warts damage. HPV protein positive superficial epithelial cells of the genital warts can appear a reddish weak positive reaction.
Third, histochemical examination
Take a small amount of lesions organizations made smears stained with specific antibodies against human papillomavirus. Such as the lesion in virus antigen, the antigen-antibody binding. Peroxidase anti-peroxidase (PAP) method endorsed is dyed red. Specificity of this method and the more rapid diagnosis.
Fourth, the pathological examination
For keratosis incomplete the spinous layer height hypertrophy, papillomatosis epidermal the sudden thickening, extend, its degree of proliferation-like pseudo-epithelial tumor-like. Stinging cells and basal cells and a considerable number of mitotic figures, resembling cancerous. However, the cells are arranged rules and clear boundaries between epithelial and dermal hyperplasia. Which is characterized by cells of the granulosa and barbed layer upper obvious vacuolization. Such vacuoles cells than normal, the cytoplasm with pale central round, deep basophilic nuclear. Usually dermal edema, telangiectasia and the surrounding dense chronic inflammatory infiltrate. Giant condyloma, Bushke-loewenstein epidermal extreme downward growth, instead of the following organizations easy mix with squamous cell, and is therefore subject to multiple biopsies. If it has a tendency to slow development, compared with a low-grade malignant process, the so-called verrucous carcinoma.
Fifth, genetic diagnosis
So far, HPV is difficult to use traditional viral culture and serological detection, the main experimental diagnostic technique of nucleic acid hybridization. PCR method developed in recent years with a specific, sensitive, simple, rapid, and opened up new avenues for HPV testing.
(A) the acquisition and processing of specimens
1. Acquisition and pre-processing of the specimen: scraper or saline infiltration swab taken from the vagina and cervix, mouth secretions and cells. While for cytological examination, the specimen is placed in 5ml containing 0.05% thimerosal in PBS, washed 2 times with PBS and centrifuged (3000g, 10min), the deposition the resuspended 1mlPBS in, take 0.5ml cell suspension DNA extraction .
2. Specimen nucleic acid extraction: 1 volume of cell suspension plus 10 times the volume of cell lysis buffer (10mmol / L Tris-HCl, pH 7.4,10 mmol / L EDTA, 150mmol / L NaCl, 0.4% SDS, 1.0mg/ml proteinase K ) overnight at 37 ° C under processing;, and an equal volume of phenol / chloroform (1:1), chloroform / isoamyl alcohol (24:1) and extracted two times each; plus 1/10 volume of 3mol / L NaAc (pH 5.2) and 2.5 times the volume of absolute ethanol is set to -20 ° C 2 h or overnight precipitation DNA; plus 1 volume of ethanol and dried 1; RNA enzyme-containing (100μg/ml) with 60μl of TE solution (10mmol / L Tris-HCl, pH 8.0,1.0 mmol / L EDTA) dissolved DNA, 37 ℃ incubated for 30min.
(B) PCR amplification
1. Primer design and synthesis: HPV genome can be divided into the early region (E) and late zone (L), each zone containing a series of open reading frame the frameworks (ORF). Sequence analysis showed that conserved sequences are non-coding regions of the various types of HPV E1, E6, E7 and L1 region. Manos choose from HPVL1 District conserved sequences designed synthetic primers MY11 and MY09 in Table 1, the primers and HPV 6,11,16,18 and 33-type complementary sequences could be amplified type HPV.
The Manos and design HPVL1 universal primers
2. PCR reaction reagents: Taq DNA polymerase (2U/ml), 10mmol / L mM dNTP stock solution (dATP, dCTP, dGTP, dTTP each 10mmol / L), 10 × PCR buffer (500 mmol KCl, 40 mmol / L MgCl2, 100 mmol / L Tris-HCl, pH 8.5), 100μmol / L MY11 and MY09 stock solution sterilized glass distillation prepared in distilled water.
3. PCR amplification methods and procedures: The 100 μl PCR reaction solution ‘with a sterile 0.5ml silicide plastic centrifuge tube amplification reaction is performed for the reaction tube.
(1) the preparation of pre-mixed reagents and repackaging before the experiment. The premixed reaction reagent including the addition to the sample DNA outside of the other kinds of PCR reaction reagents.
(2) each reaction tube were successively added 10μl the specimens and 90μl premixed reagents.
(3) was added 80-100μl paraffin oil, quick spin on a desktop centrifuge for a few seconds, so that each of the reaction reagent collected in a reservoir under. Currently, PCR reagents, have been commercialized, the reaction volume of 25μl. The added sample DNA can only use.
(4) the reaction tube set PCR amplification cycle parameters 95 ° C for 30s at 55 ° C 40s, 72 ° C 50s cycle 35 times, 72 ℃ for 5min.
4. Positive and negative controls for each test should be set. The recombinant plasmid containing the HPV (per reaction 100pg) or cell lines (such as lines Caski, HeLa) containing HPV DNA as positive controls, as a negative control in the human cell line DNA of HPV.
(C) detection and analysis of the amplification product
1. Gel electrophoresis: the amplification after the end of the reaction, remove the reaction tube, the mixture was cooled to room temperature, taken 10μl PCR product was purified by 5% -7% polyacrylamide gels or 1.5% agarose gel electrophoresis, ethidium bromide staining, ultraviolet analyzer under analysis, and a molecular weight of approximately 450bp at obvious DNA band.
2. Nucleic acid hybridization: If no clear gel electrophoresis of DNA or a need to determine the specificity of the DNA band the common mixed probes available tags, and (or) the type-specific probes for Southern blot hybridization, dot blot hybridization verification.
System 32P ATP labeled oligonucleotide probe according to standard methods, required to achieve a specific activity of approximately 107cpm/pmol. The hybridization solution containing 2 × 106-5 × 106cpm probe / ml. 2-3h, followed by hybridization in 55 ℃ slow shaking at 30-55 ° C with a washing solution (2 × SSC, 0.1% SDS) rinse quickly hybridization membrane, removing excess probe. And then washing the membrane, its conditions, depending on the nature of the probe varies: public mixed probes, 55 ℃ to wash membrane 10min; MY12, MY13 and MY16 probe ,56-57 ℃ for 10 min, and the medium was changed to wash; MY14 WD74 probe ,58-59 ℃ 10min, the medium was changed to wash.
PCR method for detection of HPV superior to nucleic acid hybridization method. Its high sensitivity, to the direct analysis of the gel electrophoresis results of the GP-PCR method, can be detected in samples of 200 copies of HPV DNA, the terms of nucleic acid hybridization, PCR product, enhanced sensitivity can be detected 10 copies of HPV DNA .
In view of the high sensitivity of the PCR technique, exfoliated cells of the genital tract samples is sufficient to meet the test requirements, to avoid a biopsy in grinding Organization complicated. In normal circumstances, the PCR amplification product by gel electrophoresis, the DNA can be directly observed generated to make the diagnosis. Therefore, PCR technology to detect HPV experimental period is short, simple and fast.