abstract
This disease is also called condyloma acuminatum, genital warts or venereal warts. It is a sexually transmitted disease caused by human papillomavirus (HPV) infection. There are many types of human papillomavirus, among which HPV 1, 2, 6, 1 1, 16, 18, 3 1, 33, 35 are the main types, among which HPV1.
diagnose
1. History of unclean sex.
2. Typical lesions are papules, papillae, cauliflower-like or cockscomb-like fleshy vegetation in wet parts such as genitals or perianal regions, with rough and keratinized surfaces.
3. Acetic acid white test was positive, and pathological sections showed poor keratinization and hollow cells.
4. HPV -DNA related sequences can be detected by nucleic acid hybridization, and specific HPV -DNA amplification bands can be detected by PCR.
Treatment measures
Because there are no specific antiviral drugs at present, the treatment of condyloma acuminatum must adopt comprehensive treatment.
(a) treatment incentives: (excessive leucorrhea, too long foreskin, gonorrhea).
(2) improve the body's immunity.
(3) Application of disease-resistant drugs. Generally, it can be cured as long as the law of comprehensive treatment is adhered to.
1. Surgical treatment
For single condyloma with small area, it can be surgically removed; For the huge condyloma acuminatum, Mohs operation can be used to remove it, and frozen sections are used to check whether the injury is removed cleanly during the operation.
2. Cryotherapy
Low temperature liquid nitrogen (-196℃) was used to treat condyloma acuminatum by pressure freezing to promote the necrosis and shedding of wart tissue. This method is suitable for condyloma acuminatum with a small number and area. 1-2 treatments are feasible, with an interval of one week.
3. Laser therapy
CO2 laser is usually used to treat condyloma acuminatum by cauterization. This therapy is most suitable for condyloma acuminatum in vulva, penis or perianal region. Single or a small number of multiple condyloma can be treated once, and multiple or large condyloma can be treated for 2-3 times, and the interval is usually one week.
4. electrocautery therapy
High-frequency electroacupuncture or electrotome was used to remove condyloma acuminatum. Methods: Electrocautery was performed after local anesthesia. This therapy is suitable for condyloma acuminatum with small quantity and area.
5. Microwave therapy
Using microwave surgical treatment machine and lidocaine local anesthesia, insert the tip of the rod-shaped radiation probe into the base of the wart, and when the wart becomes smaller, darker in color and turns from soft to hard, complete radiation curing, and then pull out the probe. Coagulated lesions can be removed with tweezers. In order to prevent recurrence, the remaining base part can be solidified again.
6. Beta-ray therapy
We have achieved satisfactory results in the treatment of condyloma acuminatum with β-ray. This method has the advantages of high curative effect, no pain, no injury, few side effects and low recurrence rate, which is worth popularizing in clinic.
7. Drug therapy
(1) Plantago asiatica: This therapy is suitable for condyloma acuminatum in damp parts, such as glans penis and perineum, where the foreskin is too long and uncircumcised. But condyloma acuminatum can't be treated with podophyllum. Before applying 20% turpentine tincture to the focus or taking medicine, protect normal skin or mucosa around the focus with oily antibacterial ointment, then apply medicine, rinse with 30% boric acid water or soapy water for 4-6 hours after use, and repeat after 3 days if necessary. This medicine is the first medicine used to treat this disease abroad, and it can be cured at one time. However, it has many disadvantages, causing great damage to tissues, and improper use will cause local ulcers. Toxic, mainly manifested as nausea, intestinal obstruction, leukopenia and thrombocytopenia, tachycardia, urinary incontinence or oliguria, so it must be used with caution. When the above reaction is found, the drug should be stopped immediately.
(2) Antiviral drugs: 5% phthalamide cream or 0.25% herpes cream can be used for external use, twice a day. Oral acyclovir, 200mg each time, 5 times a day, or external use of its ointment, injection of interferon-α, 3 million units a day, 5 days a week. Or interferon 3 million units injected into the wart base twice a week. 2-3 weeks, the main side effect is influenza-like syndrome, and the side effects of local medication are less and lighter.
(3) Corrosive agent or disinfectant: 30%-50% trichloroacetic acid or saturated dichloroacetic acid or 18% peracetic acid is commonly used. The mixed solution of 10% salicylic acid glacial acetic acid or 40% formaldehyde, 2% liquefied phenol and 75% ethanol distilled water is applied to the glans penis and perianal condyloma once a day or every other day, and the effect is very good. Disinfectant can be 20% tincture of iodine for external use, or 2.5-5% tincture of iodine can be injected into the wart base, with 0.1-kloc-0/.5ml each time, or bromogeramine or 0. 1-0.2% for external use, and the latter should be combined with systemic treatment.
(4) Anticancer drugs
① 5- fluorouracil (5-F u): Generally, 5% ointment or cream is used externally, twice a day, and 3 weeks is a course of treatment. Wet compress with 2.5%~5% fluorouracil was used to treat condyloma acuminatum around penis and anus for 20 minutes each time,/kloc-0 times a day, and 6 times was 1 course of treatment. Polyethylene glycol can also be used as matrix, and 5- F u powder accounting for 5% of its dry matter can be added to make suppository for the treatment of condyloma acuminatum in male and female urethra, or 5- F u can be injected into the matrix, and various kinds can be injected in batches.
② Taitepai: It is mainly used for condyloma acuminatum in urethra which is ineffective in 5-F u treatment. Suppositories (each containing15 mg) are used for 8 days every day, or 60mg of this product can be added to 10- 15ml sterile water and dripped into urethra for half an hour every week, which has side effects of urethritis. You can also use this product 10mg plus 10ml to soak the affected area for three times a day, each time for half an hour, to treat condyloma acuminatum of penis and condyloma of glans penis, mainly for other patients with residual or recurrent warts after treatment. You can also dilute this solution twice and soak it locally to prevent recurrence.
Colchicine: 2-8% physiological saline solution is applied externally, divided into two times, with an interval of 72 hours, to treat condyloma acuminatum of penis, and superficial erosion may occur after application.
④ Enrique
Firstly, by competing with herpesvirus, Annlic consumes the polymerase needed for herpesvirus replication, thus making herpesvirus lose the basic substances needed for reproduction, making herpesvirus unable to replicate in vivo and eventually dying completely.
Secondly, Annlic not only has good long-term efficacy and safety, but also has excellent drug resistance. The clinical research results of more than 18000 cases of genital patients in the world show that Annlic-ALICK can kill the virus quickly and effectively. After 15 days of treatment, the number of viruses in serum decreased by about 1000 times, thus significantly improving the key indexes of liver histology, biochemistry and serology. In addition, long-term treatment with Enrique will gradually improve the curative effect. After 1-2 months of continuous treatment, about 99% of patients have been unable to detect the DNA (deoxyribonucleic acid) of herpes condyloma virus. In addition, the long-term treatment of Enrique has good safety, no adverse events and no side effects on human body.
In the treatment of herpetic condyloma acuminatum, drug resistance is rare and appears late, and the long-term treatment effect is no longer troubled by virus drug resistance, which makes both doctors and patients more confident in the treatment. Furthermore, there is no cross-resistance between Annlic and other antiviral drugs, that is to say, even if a few patients take antiviral drugs for a long time, Annlic still has absolute efficacy.
Thirdly, Annlic has effects on various herpes viruses and condyloma acuminatum, including patients with genital herpes and herpes simplex, patients with initial treatment and retreatment, and patients with acyclovir resistance. These characteristics of Enrique can help patients achieve the therapeutic goal of chronic reproductive virus infection, thus becoming a brand-new choice for first-line antiviral treatment of chronic reproductive virus infection.
The fourth feature is that Annlic is easy to use, as long as it is sprayed.
There are many kinds of anti-herpes and condyloma acuminatum drugs, and their treatment mechanisms are different. Improper use will increase the burden on the liver and should be used selectively. While applying antiviral drugs, we should also find out the side effects that lead to poor liver and kidney function, treat both the symptoms and root causes, and achieve the purpose of protecting health and eliminating viruses in the body.
8. Immunotherapy
① Autovaccine method: Homogenize the patient's own wart tissue (melt the cold to inactivate the virus), heat it (56℃ for one hour) and take the supernatant for injection, which can be used for intractable perianal condyloma acuminatum.
② Interferon inducer: Polymyocytes and Tyrone can be used. Polymyocytes were injected with 2 ml every day for 10 days, and continued to be used after stopping 1-2 months. Tirolone 300 mg, 3 times a day, 4 days off, or 600 mg every other day.
③ Interferon, BCG-Ⅱ, Lingmycin and Libaiduo were used in combination, and the curative effect was good.
aetiology
Human papillomavirus (HPV) causes warts on the skin, and forms proliferative lesions in pharynx, perianal region and genital mucosa. Its virus type is small DNA virus. Most of the lesions caused by human papillomavirus infection are benign and can subside on their own, but there are also cases of deterioration. For example, squamous cell carcinoma has been reported on perianal and genital mucosa. There is also a rare hereditary skin disease, that is, skin cancer secondary to verrucous epidermal dysplasia (EV), and human papillomavirus has been detected in cancer cells.
Human papillomavirus (HPV) is a member of genus A of Mammotoviridae, and its outer shell consists of icosahedron with a diameter of 50-55nm and circular double-stranded DNA with 7900 base pairs. The size and shape of virus particles are very similar to those of oral polyoma virus under electron microscope. Papillomavirus (PV) is species-specific, and human papillomavirus cannot be propagated in tissue culture or experimental animal models.
The structural protein composition of the virus: 85% PV particles, SDS——PAGE shows that the virus has the main capsid protein with molecular weight of 56.000; The secondary capsid protein with molecular weight shifted to 76.000 found that four cell histones were related to viral DNA.
The genome structure of all human papillomavirus viruses is similar. According to the procedure of DNA hybridization under strict conditions, the virus type and subtype can be determined. Only 50% of different types of human papillomavirus hybridize with other types of viral DNA. Up to now, more than 60 kinds of human papillomavirus have been found. With the deepening of research, more new types of human papillomavirus will be found.
pathogenesis
Human papillomavirus infection of condyloma acuminatum is transmitted through sexual contact, and minor trauma at the contact site can promote infection. Three kinds of squamous epithelium (skin, mucosa and metaplasia) are all sensitive to human papillomavirus infection. Each type of human papillomavirus is related to special clinical damage, and it has its own predilection site for skin or mucosal squamous epithelium. When exfoliated surface cells or keratin fragments containing a large number of virus particles enter the cracks of susceptible epithelium, infection may occur, which may be caused by direct contact or rare automatic inoculation or contaminated underwear, bathtub, bath towel and bedpan.
After the virus infects the human body, it can lurk between basal keratinocytes and replicate in the epidermal cell layer. Human papillomavirus invades the nucleus, causing cells to divide rapidly, and at the same time, with the proliferation and diffusion of virus particles, characteristic papilloma is formed. When the structural polypeptide is expressed in the later stage, structural protein assembly particles appear. The virus is mainly concentrated in the nucleus of granular layer, and there are many hollow cells in granular layer of epidermis. Histologically normal epithelial cells also have human papillomavirus, and the residual DNA after treatment often leads to the recurrence of the disease.
The host immune response to human papillomavirus infection includes cellular immunity and humoral immunity.
First, cellular immunity
The cellular immune state of human body is one of the important foundations that affect the occurrence and prognosis of condyloma acuminatum. Cellular immunity is more important than humoral immunity. Clinically, the skin rash of patients with condyloma acuminatum with cellular immune deficiency often persists, the number of inhibitory T cells in peripheral blood increases, the function of NK cells is low, and the production of r- interferon and interleukin -2 decreases. However, the infiltration of activated T cells and NK cells often appears in the subsided wart lesions, and some keratinocytes are HLA-DR positive.
With immunosuppression or immunodeficiency, the incidence of genital human papillomavirus infection and human papillomavirus-related diseases increases. In condyloma acuminatum, helper T cells are depleted, and the CD4/CD8 ratio is inverted, and its value is < 1. In the peripheral blood of CA patients, the percentage of inhibitory/cytotoxic T cells increased significantly, while the proportion of helper/inducible T cells and helper/inhibitory T cells decreased. Langerhans cells in cervical CA and cervical intraepithelial neoplasia (CIN) decreased significantly. The ability of NK cells to produce γ -interferon and interleukin -2 decreased in patients with CA. The cytolytic activity of NK cells on keratinocytes containing human papillomavirus-16 in patients with acinetobacter baumannii papulosis and anorectal cancer decreased, which may be caused by the defect of recognizing disease-specific target cells. Keratinocytes in cervical CA do not express MHCⅱⅱ antigen (HLA-DR), and the lack of this antigen presenting function will destroy the immune monitoring function.
Second, humoral immunity.
At present, the results of serological tests show that: ① the production rate of anti-late protein antibody is 25%-65%, which is higher than that of anti-early protein antibody; ② The detected human papillomavirus antibody seems to be type-specific, and there is no cross reaction; ③ Anti-human papillomavirus-16E7 antibody is closely related to the existence of cervical cancer; ④ Anti-human papillomavirus-16E4 antibody is also a sign of cervical cancer, recurrence or recent human papillomavirus infection; ⑤ It is estimated that the positive rate of IgG antibody produced by adults and children is the same, and the positive rate of different types is between 10%-75%; ⑥ Only 50%-70% of patients with human papillomavirus-16 or 18 tumor antibodies can be detected.
Third, condyloma acuminatum naturally subsided.
At present, there is no systematic evaluation on the natural regression of CA. However, the placebo-controlled study found that the natural regression rate was between 0%, 17%, 18% and 69%. After CA subsided or was cured, 45% patients still had latent infection, and 67% of them relapsed.
epidemiology
I. Prevalence
Condyloma acuminatum is caused by human papillomavirus. At present, it is recognized that the disease is on the rise and has become the most common disease among sexually transmitted diseases, and the prevalence rate of young adults can reach 0.5%- 1%. The incidence of condyloma acuminatum in Britain increased from 30/65438+ million in 1970 to 260/65438+ million in 1988, almost eight times, and in the United States it increased six times from 1966 to 1984. Similar to AIDS, symptomatic condyloma acuminatum only represents the top of the infected iceberg, so if subclinical infection is considered, human papillomavirus infection may be the most common sexually transmitted disease. The modes of transmission of this infection include direct transmission and indirect transmission, but sexual contact is the most common, and the closer the damage, the more contagious it is. It is estimated that there is a 50% possibility of one-time contact infection. Followed by direct non-sexual contact, such as self-infection and neonatal infection through the birth canal; Followed by indirect contact, it is possible to infect and speculate through pollution, but the virus cannot be cultivated and diagnosed.
Second, the risk factors of condyloma acuminatum
(1) Sexual behavior: The number of sexual partners and premature sexual intercourse are the factors leading to human papillomavirus infection.
(2) Immunosuppression: HPV infection and HPV-related cancer seem to be the late complications of chronic immunosuppression. Renal transplant recipients are at increased risk of developing CA.
(3) HIV infection: The probability of HPV infection and HPV-related tumors increases.
(4) Age and pregnancy: The peak of the prevalence of human papillomavirus in gynecological smears is 20-40 years old, and the prevalence rate decreases steadily with age; The detection rate of human papillomavirus was high during pregnancy, but decreased after delivery.
Criteria for diagnosis and treatment of condyloma acuminatum
1. Diagnostic criteria
The contact history of 1. 1 includes unmarried contact history, spouse infection history or indirect infection history.
Clinical manifestations of 1.2
1.2. 1 Men and women occasionally appear multiple pink, grayish white or grayish brown papules or nipple, comb or cauliflower-like vegetation around genitals, perineum or anus. A few giant condyloma acuminatum with papilloma-like hyperplasia, that is, Brucella-Loeb giant condyloma acuminatum.
1.2.2 Symptoms include itching, foreign body sensation, pressure or pain, increased brittleness of skin lesions and frequent bleeding. Women can have increased leucorrhea.
1.2.3 Women suffering from condyloma acuminatum should be differentiated from pseudo condyloma (villous labia minora), while men should be differentiated from pearl-like penile papules. Men and women should also be differentiated from verruca plana, bowenoid papulosis and genital syringoma.
1.2.4 Apply 5% acetic acid solution to the affected area, and it will turn white after 3 ~ 5 minutes.
1.3 Laboratory inspection
1.3. 1 Histopathological changes of characteristic hollow cells infected with human papillomavirus in skin biopsy.
1.3.2 When necessary, detect human papillomavirus by skin biopsy antigen or nucleic acid. Common ones are HPV6, 1 1, and rare ones are HPV 16, 18.
1.4 case classification
1.4. 1, with indicators of 1. 1 and 1.2.
1.4.2 The confirmed cases have any of the indicators in 1. 1, 1.2, 1.3.
2. Handling principle
2. 1 handling principle
2. 1. 1 Pay attention to whether the patient is infected with gonococcus, chlamydia, mycoplasma, trichomonas, mold and other pathogens at the same time, and if so, treat them at the same time.
2. 1.2 if the spouse and sexual partner of the patient have condyloma acuminatum or other sexually transmitted diseases, they should be treated at the same time.
2. 1.3 Avoid sexual life during treatment.
2.2 Judgment and prognosis
The standard of healing is to remove the hyperplastic wart. The prognosis of condyloma acuminatum is generally good, but the recurrence rate is high.
2.3 Management and prevention
2.3. 1 condyloma acuminatum should be reported only once per patient. Patients who have not been diagnosed in the past should be reported at the first diagnosis.
2.3.2 Patients with condyloma acuminatum should avoid sexual life or use condoms to prevent transmission to their spouses.
2.3.3 Patients should receive treatment as soon as possible.
2.3.4 Strictly observing monogamy is the best way to prevent this disease.
clinical picture
The incubation period is 3 weeks to 8 months, with an average of 3 months, which is more common in sexually active young men and women. Male and female patients with a peak age of 20-25 years and an average course of 3-5 months will get sick soon after sexual contact, while male patients with an average course of 12 months may not get sick. Most patients are generally asymptomatic. The damage varies in size and shape. It can be only a few, or it can be as big as most needles: it can grow into a large tumor-like object in vulva and anus, which has a sense of oppression; Have an unpleasant smell; Sometimes small condyloma can cause pudendal itching and discomfort, and patients may have hematuria and dysuria; Condyloma acuminatum in rectum can cause pain and bloody stool, while condyloma acuminatum in rectum can cause urgency and then heaviness.
Male patients are prone to prepuce frenulum, coronary sulcus, prepuce, urethra, penis, perianal region and scrotum. At the initial stage of the disease, it is reddish or dirty red millet-like vegetation, which is soft and slightly pointed at the top, and gradually grows or increases. It can develop into a papillary or cystic shape, with a slightly wider or banded bottom and particles on the surface. It often grows in the anus, looks like cauliflower, and the surface is wet or bleeding. There is often pus between particles, which gives off a stench and can be infected after scratching. Genital warts located in dry parts with low humidity are usually small and flat. Warts located in hot and humid areas are often filamentous or papilloma-like, which are easy to fuse into large pieces. Patients with severe liver disease may have enlarged condyloma. Pregnancy can make condyloma acuminatum relapse or accelerate its growth.
Sub-clinical infection refers to a pathological change that can't be recognized by naked eyes in clinic, but it can turn white by local application or wet application of 3%-5% acetic acid solution for 5- 10 minutes in the infected area of human papillomavirus, which is called "white acetate phenomenon".
Human papillomavirus infection and tumorigenesis;
(A) Human papillomavirus is related to the occurrence of skin tumors.
It seems to play a decisive role in skin cancer and other anatomical tumors. Human papillomavirus-1 1, 16, 18 DNA can be found in oral benign vegetation and precancerous lesions. It is reported that human papillomavirus -6 papilloma in larynx has transformed into laryngeal cancer, and epidermodysplasia verrucosa (EV) is evidence of the potential carcinogenesis of human papillomavirus. Many human papillomavirus DNA types were found in enterovirus lesions. Human Papillomavirus -5, Human Papillomavirus -8, Human Papillomavirus-14, Human Papillomavirus-17 and Human Papillomavirus -20 were detected in patients' skin squamous cell carcinoma, which seemed to be malignant caused by pre-existing virus damage.
(2) Condyloma Acuminatum and anal cancer.
Genital cancer is related to the type of human papillomavirus. Human papillomavirus -6, 1 1, 16, 18 were found in genital cancer tissues by DNA hybridization.
1. Cervical cancer: According to the relationship between HPV and cervical cancer, there are two types: low-risk type mainly refers to HPV -6, 1 1, and high-risk type refers to HPV-16, 18. Gissmann et al. observed 57.4% invasive cervical cancer. Others isolated human papillomavirus -33 and human papillomavirus -35 from invasive cervical cancer.
2. Squamous cell carcinoma of skin (SCC): CA caused by human papillomavirus infection may also be precancerous damage, and may develop into anal genital SCC, which indicates that human papillomavirus is an important factor of vulva, penis and anal genital SCC. CA, giant CA and verrucous SCC constitute the spectrum of genital precancerous lesions and cancer damage. Some cases of genital cancer have CA in the surrounding skin, and sometimes it is a typical CA with naked eyes, but histological examination found isolated lesions of SCC.
3. Bowenoid papulosis: it is common around penis, vulva or anus, and human papillomavirus-16 DNA has been found in skin lesions.
accessory examination
First, white acetic acid test.
When the wart is coated with 3-5% acetic acid for 2-5 minutes, the focus turns white and slightly bulges, and anal lesions may take 15 minutes. The principle of this experiment is the result of protein and acid condensation whitening. Keratin produced by human papillomavirus infected cells is different from that produced by normal uninfected epithelial cells, and only the former can be decolorized by acetic acid. Acetic acid white test is highly sensitive to detect human papillomavirus, which is superior to conventional test to observe histological changes. However, in the case of epithelial thickening or traumatic abrasion, false positive and false positive albinism occasionally appear, which is unclear and irregular. The CDC suggested that the white acetate test is not a specific test, and false positives are more common.
II. Immunohistochemical examination
Peroxidase-anti-peroxidase method (PAP) is often used to display the virus protein in condyloma, so as to prove the existence of virus antigen in warts. When human papillomavirus protein is positive, the superficial epithelial cells of condyloma acuminatum can have reddish positive reaction.
Three. Histochemical examination
A small amount of diseased tissues were made into smears and stained with specific antibodies against human papillomavirus. If there is a virus antigen in the lesion, the antigen and antibody bind. In the peroxidase anti-peroxidase (PAP) method, the nucleus can be dyed red. This method is specific, rapid and helpful for diagnosis.
Four, pathological examination
The main reasons are poor keratinization, high spinous process hypertrophy, papilloma-like hyperplasia, thickening and lengthening of epidermal process, and its proliferation degree can be similar to pseudoepithelioma. There is considerable mitosis between spinous cells and basal cells, which seems to be canceration. But the cells are arranged regularly, and the boundary between hyperplastic epithelium and dermis is clear. It is characterized by obvious vacuole formation in the upper cells of granular layer and prickle layer. This vacuolar cell is larger than normal cells, with pale cytoplasm and a large round deep basophilic nucleus in the center. Usually dermal edema, telangiectasia and dense chronic inflammatory infiltration around. Bushke-loewenstein's huge condyloma acuminatum grows extremely downward, replacing the underlying tissue, which is easy to mix with squamous cells and needs multiple biopsies. If there is a slow development trend, it is a process of low-grade malignant transformation, called verrucous cancer.
Verb (abbreviation of verb) gene diagnosis
Up to now, traditional virus culture and serological techniques are difficult to detect human papillomavirus, and the main experimental diagnostic technique is nucleic acid hybridization. The PCR method developed in recent years has the advantages of specificity, sensitivity, simplicity and rapidity, which opens up a new way for the detection of human papillomavirus.
(1) specimen collection and processing
1. Specimen collection and pretreatment: Take secretions and cells from vaginal and cervical external orifice with scraper or cotton swab soaked in normal saline. At the same time of cytological examination, the specimen was put into 5ml PBS containing 0.05% thiomersal, washed twice by PBS centrifugation (3000g, 10min), the precipitated cells were resuspended in 1mlPBS, and DNA was extracted from 0.5ml cell suspension.
2. Extracting nucleic acid from the sample: according to the cell suspension of 1 volume, add cell lysate of 10 volume (10mmol/L Tris-HCl, pH 7.4, 10mmol/L EDTA, 150mmol/L NaCl. Extracting twice with the same volume of phenol/chloroform (1∶ 1) and chloroform/isoamyl alcohol (24∶ 1) respectively; Add110 volume of 3mol/L NaAc(pH 5.2) and 2.5 times the volume of anhydrous ethanol, and let it stand at -20℃ for 2 hours or overnight to precipitate DNA；; Washing with 1 volume of ethanol 1 time; Dissolve DNA in 60μl of TE solution containing ribonuclease (100μg/ml Tris-HCl, pH 8.0, 1.0mmol/L EDTA) and incubate at 37℃ for 30 minutes.
(2) PCR amplification
1. Primer design and synthesis: The genome of human papillomavirus can be divided into early region (E) and late region (L), and each region contains a series of open reading frames (ORFs). Sequence analysis showed that all types of human papillomavirus had conserved sequences in non-coding region and E 1, E6, E7 and L 1 regions. Manos et al. designed and synthesized primers MY 1 1 and MY09 from the conserved sequence of HPVL 1, as shown in table 1. This primer has complementary sequences with human papillomavirus 6, 1 1, 16, 18 and 33, and can also amplify other primers.
2.PCR reagents: Taq DNA polymerase (2U/ml) and 10mmol/L dNTP stock solution (dATP, dCTP, dGTP and dTTP are 10mmol/L respectively). 10×PCR buffer (500mmol KCl, 40mmol/L MgCl2, 100mmol/L Tris-HCl, pH 8.5),100 μ mol/l my1and MY09 stock solution.
3.PCR amplification method and procedure: use 100μl PCR reaction solution, and use 0.5ml sterile silicified plastic centrifuge tube as the reaction tube for amplification reaction.
(1) Pre-mixed reagents are prepared and packaged before the experiment. Premixed reagents include all types of PCR reagents except sample DNA.
(2) Add 10μl sample and 90μl premixed reagent to each reaction tube in turn.
(3) Add 80- 100μl paraffin oil, centrifuge quickly for a few seconds on a desktop centrifuge, and collect all the reaction reagents under the oil layer. At present, PCR reagents have been commercialized, and the reaction volume is 25 μ L, and only sample DNA can be added when used.
(4) Put the reaction tube on a PCR amplifier, with the circulating parameters of 95℃ for 30s, 55℃ for 40s, 72℃ for 50s, and finally extend it at 72℃ for 5min.
4. Positive and negative controls should be set for each test. The recombinant plasmid carrying human papillomavirus (100 pg/ reaction) or cell line DNA containing human papillomavirus (such as Caski and HeLa) were used as positive control, while the cell line DNA without human papillomavirus was used as negative control.
(3) Detection and analysis of amplified products
1. gel electrophoresis: after the amplification reaction, take out the reaction tube, cool it to room temperature, take 10μl amplification product, electrophoresis it with 5%-7% polyacrylamide gel or 1.5% agarose, staining it with ethidium bromide, and uv analyzer analysis. The results showed that there were obvious DNA bands at the molecular weight of about 450bp.
2. Nucleic acid hybridization: If there is no definite DNA in gel electrophoresis or the specificity of DNA bands needs to be determined, Southern blot hybridization and dot hybridization can be verified by using labeled public mixed probes and/or type-specific probes.
According to the standard method, 32P ATP labeled oligonucleotide probes need to achieve a specific activity of about 107cpm/pmol. The hybridization solution should contain 2× 106-5× 106cpm probe /ml. Hybridization was carried out at 55℃ for 2-3h, and then the hybridization membrane was quickly washed with washing solution (2×SSC, 0. 1%SDS) at 30-55℃ to remove redundant probes. Then the membrane was washed, and the conditions were different according to the different probes used: common mixed probe, membrane washing at 55℃ for 65438±00min；; MY 12, MY 13 and MY 16 probes, at 56-57℃ 10 min, and washed again 1 time; MY 14 and WD74 probes, at 58-59℃ 10min, are also changed and washed 1 time.
Detection of human papillomavirus by PCR is superior to nucleic acid hybridization. Its sensitivity is very high. GP-PCR can directly analyze the results by gel electrophoresis, and can detect 200 copies of human papillomavirus DNA. If the PCR product is detected by nucleic acid hybridization, the sensitivity is improved, and the 10 copy of human papillomavirus DNA can be detected.
In view of the high sensitivity of PCR technology, it is enough to take exfoliated cells of reproductive tract as detection materials to meet the detection requirements, avoiding the complicated operations of taking samples for biopsy and grinding tissues. In general, PCR amplification products can be directly diagnosed by gel electrophoresis and observing the generated DNA. Therefore, PCR technology is rapid and simple for the detection of human papillomavirus.
prevent
Controlling sexually transmitted diseases is the best way to prevent condyloma acuminatum. Discover and treat patients and their sexual partners; Carry out health education and control sexual behavior; Condoms can prevent human papillomavirus infection, and there is no effective vaccine at present.
Doing the following can effectively prevent condyloma acuminatum:
1. Resolutely put an end to promiscuity: 60% of patients with condyloma acuminatum are infected through sexual contact. One member of the family gets sick from society, but also spreads it to his spouse through sexual life, and may spread it to other family members through close life contact, which not only brings physical pain, but also causes family discord and mental stress. Therefore, improving sexual morality and not having sex outside marriage is an important aspect to prevent condyloma acuminatum.
2. Prevent contact infection: Don't use other people's underwear, swimsuits and bathtubs; Don't wash basins and pools in public baths; Advocate shower; Don't take a shower directly in the chair after taking a shower. Try to use squatting toilets in public toilets; Wash your hands with soap before going to the toilet; Don't swim in a swimming pool with high density and poor disinfection.
3. Pay attention to personal hygiene: clean the vulva every day, change underwear, and clean personal underwear separately. Even among family members, one person should share a washbasin and towel.
4. Sexual life should be forbidden after the spouse is ill. If the spouse only receives physical therapy, although the condyloma acuminatum visible in the vulva disappears, the patient still carries human papillomavirus, and should also receive comprehensive treatment of oral drugs and external washing drugs, and review after treatment. If you have sex during this period, you can use condoms for protection.
5. Pregnant women with condyloma acuminatum can choose cesarean section in order to avoid infecting the fetus during delivery. Don't bathe in the same basin with your baby after childbirth.