What are the examination and diagnosis methods for genital warts?

 　　Genital warts are an epithelioma-like growth caused by infection with the human papillomavirus (HPV). The diagnosis of genital warts is mostly based on the characteristics of the rash, location of onset, development, and possible contact history. However, a few patients need to undergo some auxiliary experiments to confirm the diagnosis. Specific experimental diagnostic methods are introduced as follows:

　　1. Acetic acid whitening test

　　Apply 3-5% acetic acid externally to the wart for 2-5 minutes. The lesion will turn white and slightly swell. It may take 15 minutes for anal lesions. The principle of this test is that the protein and acid coagulate and turn white. The keratin produced by HPV-infected cells is different from that produced by normal uninfected epithelial cells. Only the former can be decolored by acetic acid. The acetic acid white test has a high sensitivity for detecting HPV, and it is better than conventional testing for observing histological changes. However, occasionally false positives appear in cases of epithelial thickening or traumatic abrasion, and false positive whitening signs appear unclear and irregular. The US CDC reminds that the acetic acid white test is not a specific test and false positives are common.

　　2. Immunohistological examination

　　The peroxidase-anti-peroxidase method (PAP) is commonly used to display viral proteins in genital warts to prove that there are viral antigens in the wart lesions. When HPV protein is positive, a light red weak positive reaction may appear in the superficial epithelial cells of genital warts.

　　3. Histochemical examination

　　Take a small amount of the damaged tissue to make a smear and stain it with an antibody specific against human papillomavirus. If there is a viral antigen in the lesion, the antigen-antibody will combine. In the peroxidase-antiperoxidase (PAP) method, nuclei can be stained red. This method is highly specific and rapid, and is helpful for diagnosis.

　　4. Pathological examination

　　The main symptoms are parakeratosis, hypertrophy of the spinous layer, papillomatous hyperplasia, thickening and elongation of epidermal processes, and the degree of hyperplasia can resemble pseudoepithelioma. Stinging cells and basal cells also have a considerable number of nuclear divisions, which is quite similar to AI-G changes. However, the cells are arranged regularly, and the boundary between the hyperplastic epithelium and the dermis is clear. It is characterized by obvious vacuole formation in the upper cells of the granulosa layer and spinous layer. This type of vacuolated cells is larger than normal, with pale cytoplasm and a large, round, deeply basophilic nucleus in the center. Typically there is dermal edema, telangiectasia, and a dense surrounding chronic inflammatory infiltrate. Bushke-loewenstein giant condyloma acuminata, the epidermis grows extremely downward, replacing the underlying tissue, and is easily mixed with squamous cells, so multiple biopsies are required. If it tends to develop slowly, it is a low-grade malignant process, which is the so-called verrucous AI-G.

　　5. Genetic diagnosis

　　So far, HPV is difficult to detect using traditional viral culture and serological techniques, and the main experimental diagnostic technique is nucleic acid hybridization. The PCR methods developed in recent years have the advantages of specificity, sensitivity, simplicity, and rapidity, opening up new ways for HPV detection.

　　(1) Collection and processing of specimens

　　1. Collection and preprocessing of specimens: Use a scraper or cotton swab soaked in physiological saline to collect secretions and cells from the vagina and external cervical os. While performing cytological examination, put the specimen into 5 ml of PBS containing 0.05% thimerosal, wash it twice with PBS centrifugation (3000g, 10 min), resuspend the sedimented cells in 1 ml of PBS, and extract 0.5 ml of the cell suspension.

　　2. Extraction of sample nucleic acid: Add 1 volume of cell suspension to 10 times the volume of cell lysis buffer (10mmol/L Tris-HCl, pH 7.4, 10mmol/L EDTA, 150mmol/L NaCl, 0.4% SDS, 1.0mg/ml Proteinase K) was treated overnight at 37°C; and equal volumes of phenol/chloroform (1:1) and chloroform/isoamyl alcohol (24:1) were extracted twice each; add 1/10 volume of 3mol/L NaAc (pH 5.2) and 2.5 times the volume of absolute ethanol and place at -20°C Precipitate the DNA for 2 hours or overnight; add 1 volume of ethanol and wash once; dissolve the DNA with 60 μl of TE solution (10 mmol/L Tris-HCl, pH 8.0, 1.0 mmol/L EDTA) containing RNase (100 μg/ml), and incubate at 37°C.

　　(2) PCR amplification

　　1. Primer design and synthesis: The HPV genome can be divided into early region (E) and late region (L), each region contains a series of open reading frames (ORF). Sequence analysis shows that all types of HPV have conserved sequences in the non-coding regions and E1, E6, E7 and L1 regions. Manos et al. selected conserved sequences from the HPVL1 region to design and synthesize primers MY11 and MY09, as shown in Table 1. The primers have complementary sequences to HPV types 6, 11, 16, 18 and 33, and can also amplify other types.

　　2. PCR reaction reagents: Taq DNA polymerase (2U/ml), 10mmol/L dNTP stock solution (10mmol/L each of dATP, dCTP, dGTP, and dTTP), 10×PCR buffer (500mmol KCl, 40mmol/L MgCl2, 100mmol/L Tris-HCl, pH 8.5), 100μmol/L MY11 and MY09 stock solutions are distilled water prepared in sterile glass stills.

　　3. PCR amplification method and procedure: Use 100 μl PCR reaction solution and use a sterile 0.5 ml siliconized plastic centrifuge tube as the reaction tube to perform the amplification reaction.

　　(1) Prepare premixed reaction reagents before experiment and aliquot. Premixed reaction reagents include various other PCR reaction reagents except sample DNA. PCR reaction reagents contained in each reaction tube.

　　(2) Add 10 μl of specimen and 90 μl of premixed reaction reagent to each reaction tube in sequence.

　　(3) Add 80-100 μl paraffin oil and centrifuge quickly on a desktop centrifuge for a few seconds to collect each reaction reagent under the oil layer. Currently, PCR reagents are commercialized and the reaction volume is 25 μl. Only add sample DNA when using.

　　(4) Place the reaction tube on the PCR amplifier. The cycle parameters are 95°C for 30s, 55°C for 40s, and 72°C for 50s for 35 times, and finally extend at 72°C for 5 minutes.

　　4. Each test should have positive and negative controls. Use HPV-containing recombinant plasmid (100pg per reaction) or HPV-containing cell line (such as cancer ski, HeLa) DNA as a positive control, and use HPV-free human cell line DNA as a negative control.

　　(3) Detection and analysis of amplification products

　　1. Gel electrophoresis: After the amplification reaction, take out the reaction tube and cool it to room temperature. Take 10 μl of the amplification product and run it on a 5%-7% polyacrylamide gel or 1.5% agarose electrophoresis, stain it with ethidium bromide, and analyze the results with a UV analyzer. An obvious DNA band appears at a molecular weight of about 450 bp.

　　2. Nucleic acid hybridization: If there is no clear DNA in gel electrophoresis or the specificity of the DNA band needs to be determined, labeled public mixed probes and/or type-specific probes can be used for Southern blot hybridization and dot hybridization verification.

　　According to standard methods, 32P ATP-labeled oligonucleotide probes should be prepared to achieve a specific activity of approximately 107 cpm/pmol. The hybridization solution must contain 2×106-5×106cpm probe/ml. Hybridize for 2-3 hours at 55°C with slow shaking, then quickly rinse the hybridization membrane with washing solution (2×SSC, 0.1% SDS) at 30-55°C to remove excess probe. The membrane is then washed, and the conditions vary depending on the probe used: for the public mixed probe, wash the membrane at 55°C for 10 minutes; for the MY12, MY13 and MY16 probes, wash the membrane at 56-57°C for 10 minutes, and change the liquid and wash it once; for the MY14 and WD74 probes, wash the membrane for 10 minutes at 58-59°C, and change the liquid and wash it once again.

　　The detection of HPV using PCR methods is superior to nucleic acid hybridization methods. It has high sensitivity. The GP-PCR method uses gel electrophoresis to directly analyze the results and can detect 200 copies of HPV DNA in the specimen. If nucleic acid hybridization is used to detect the PCR product, the sensitivity is improved and 10 copies of HPV DNA can be detected.

　　In view of the high sensitivity of PCR technology, using exfoliated cells from the reproductive tract as the test material is sufficient to meet the test requirements, avoiding the complicated operations of biopsy sampling and tissue grinding. Under normal circumstances, the PCR amplification product is subjected to gel electrophoresis, and the resulting DNA can be observed to make a direct diagnosis. Therefore, PCR technology to detect HPV has a short experimental cycle, is simple and fast.