男性人乳头瘤病毒基因芯片检测方法的建立及临床应用
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摘要
本课题的目的是建立一种适合临床男性人乳头瘤病毒(HPV)基因亚型检测的新方法,同时选择一种易于采集、提高男性HPV阳性检出率、适合基因膜芯片法的标本类型,并对深圳计划生育门诊中男性HPV感染的流行型别和分布状况进行检测分析。
通过计算机辅助,参考经典通用引物(MY09/11)和加以改进的PGMY09/11设计23种HPV基因亚型的PCR扩增引物,根据Genbank中的HPV的型特异性序列设计及合成探针,制备可同时对18种高危型:HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 83, MM4和5种低危型:6, 11, 42, 43, 44进行分型检测的特定纸质膜芯片。
通过PCR反应,经过PCR产物与HPV膜条(探针条)杂交、显色后,HPV膜条的PC点显示蓝点才是有效膜条,根据其他蓝点显示位置判读HPV基因亚型(单一感染和多重感染)。对膜杂交结果显示单一亚型感染的阳性PCR产物纯化后进行测序。
各型HPV检测段DNA的克隆质粒进行pGEM-T载体构建。重组质粒通过T7启动子引物测序,并与该亚型HPV基因组序列进行比对,以确定构建质粒中所含的各型HPV片段与检测序列完全相同。
将经过工程菌扩增的重组质粒提纯后,应用紫外分光光度计测定核酸浓度,用Tris-HCl配制成106拷贝/ml的原始标准品,再将原始标准品稀释成不同浓度的HPV标准品,通过PCR方法、经杂交、显色后,以确定HPV分型的灵敏度。同时,检测对比男性尿道分泌物、精液及尿液三种类型标本。
得出以下结论:①本课题建立的男性人乳头瘤病毒(HPV)基因亚型检测方法具有灵敏度高、特异性好、实验程序简便、采样方便、易于设定正常对照等优点,适用于临床进行男性HPV感染的诊断,为开展相应的流行病、病因学调查提供切实可行的检测手段。②检测出的男性HPV基因亚型有:HPV-6,11,16,18,33,35,43,56和73型九种。灵敏度可达10个拷贝HPV DNA分子。③临床应用中发现深圳男性HPV检测阳性率是22.3%,男性HPV感染最常见的基因亚型是6、16和73型。就诊的男性中以31岁～40岁的年龄组最多,其次是21岁～30岁年龄组,但发现深圳男性HPV的感染与年龄无相关性。④多重HPV感染中,以双重感染33/73型感染最多,高危型和低危型混合感染最常见。⑤确定临床常规检测男性HPV感染采用尿道分泌物做为采集部位和标本具有较高的敏感性,并且采样相对简便、易行。
本研究主要创新:
同时可以检测23种HPV基因亚型是本研究的创新点,HPV基因芯片法使得临床对HPV亚型检测达到微型化、集约化和标准化,从而保证了诊断的高效、廉价、快速和简便。
Human papillomavirus (HPV)is a sexually transmitted disease widely. Over 100 different HPV types have been identified based on DNA sequence analysis, HPV can be divided into“high-risk”and“low-risk”groups on the basis of their association with oncogenic nature, approximately 60 HPV genotypes are known to infect the genital and urethral tract epithelial sites, at least 13 of which are considered“high-risk”and oncogenic. HPV are considered the important etiologic agents of cervical cancer, which have been researched much many. But they are less relatively the studies to male human papillomavirus infection than the women’s. On one hand, because the sites of physiological anatomy are different, specimens acquisition of males are very difficult than women’s, collecting shedding cells roughly. On the other hand, oncogenic genotypes of HPV to both men and women are varying degree, which in women mean bad results such as cervical cancer, et al. But detection of males infection genotypes may be vital parameter to their transforms, moreover, which are more significant to supervise fatalness of males sexual partners. Infecting HPV males are considered increasing danger to their partners, who are high-risk means of delivery. Several studies have provided estimates of HPV prevalence among men, however, these estimates range widely from 1.0% to 82.9%.Significant barriers to the synthesis and interpretation of these studies are the inconsistency of sampling techniques used, the variety and combinations of sites and specimens sampled, and the testing for different HPV genotypes, for example, the urethra, glans /corona, or scrotum samples, et al. In order to understand the epidemical condition and changing trend, there is urgent to develop an experimentation to adapt to clinical males HPV test, sampling convenient, and be able to detect high-risk and low-risk genotypes simultaneously, so that patients of infecting HPV males can be treated pertinently and epidemical surveillance.
Firstly, establishing methods
Probes of 18 high-risk subtypes and 5 low-risk subtypes were designed according to membrane DNA chip technique and Genbank data about HPV, through hybridizing, et al, 23 males HPV genotype can be detected simultaneously. Through computer, we referred to classically common primers (MY09/11) and improved them to be PGMY09/11 (5’-biotin-CGT CCM ARR GGA WAC TGA TC-3’; 5’-biotin-GCM CAG GGW CAT AAY AAT GG-3’; M=A/C,W=A/T,Y=C/T,R=A/G)as universal primers, andβ- globulin primer PC03/ GH20(5’-biotin-ACA CAA CTG TGT TCA CTA GC-3’; 5’-biotin-GAA GAG CCA AGG ACA GGT AC-3’)as majordomo genes. All primers were signed by biotins about 5’end. We designed the genotyping probes according to Genbank data associated HPV subtypes sequences, and designed the control probe according to Genbank data associatedβ- globulin sequences, all probes were signed by amino acid about 5’end. Analysis probes were fixed on special membrane to make DNA chip according order of number. Type of specimen was male urethral sample with sterile cotton swabs.HPV membrane DNA chip includes 18 high-risk subtypes: HPV-16, 18, 31, 33,35, 39, 45, 51,52, 53, 56, 58, 59, 66, 68, 73, 83, and MM4, and 5 low-risk subtypes: 6, 11, 42, 43 and 44. Through PCR reaction, and After products of PCR reaction hybridized with HPV membrane stripes (probes stripes) and show color, strips were valid when only PC blots show blue dots, meanwhile, HPV subtypes were judged with other sites blue dots on the same strips (single infection and multiple infection). Single subtype HPV infection of position PCR products was sequenced. Cloned plasmid of every HPV subtype DNA was constructed to pGEM-T carrier. Reformed plasmid was sequenced by T7 opener primer and compared with same HPV subtype genome sequence. So can confirm to the detected sequence was fully same as segment with constructed plasmid DNA. Constructed plasmid DNA after purified and amplified with engineering germs was detected nucleic acid concentration by ultraviolet analysis instrument. Original standard sample was diluted to 106 copies with Tris-HCl, which was diluted to serial HPV standard dilutions in different concentration. Sensitivity of analytic HPV genotyping can be confirmed after PCR reaction, hybridization and color display.
On account of shell protein variation of different HPV subtype, there were no cross protecting antibody among HPV subtypes basically, which can make time after time infections between high-risk and high-risk HPV subtypes, or within high-risk and high-risk. We designed primers referring to classically common primers (MY09/11) and improved them to be PGMY09/11 through computer, whose sensitivity was higher than standard MY09/11 primer. In order to avoid amplification failure for HPV primer variation in combined section, we synthesized primer with distinct alkali bases in relatively easy mutation places. Meanwhile, in order to conveniently detect amplified products with reverse hybridiza- tion, we marked one end of the improved primer with biotin and designed specific genotyping probes in HPV 23 subtypes vary section.
Experimental results demonstrate that speciality of genotyping probes can obtain distinguishing rate for one alkaline base, while we make the probes distinguishing over 30% within alkaline bases, so it is highly special the results of hybridization. Moreover, we didn’t found non-special hybridization during sequences analysis of positive samples. The sensitivity of membrane special degree of DNA chip can make ten copies DNA molecules after standard products with 23 HPV subtypes of plasmid construction were detected and show concrete subtype. HPV-6,11,16,18,33,35,43,56 and 73 were detected out , which can prove those above. The new method is suitable to be used to male clinical diagnosis, epidemical and disease reasons investigation.
Secondly, choicing adapt sample sort
Utilizing established new method, we compared three sorts of samples, HPV 23 subtypes were detected in males with comparison of urethral, seminal and urinary specimens. Our aim was to develop a right sorting of specimen for diagnosis of human papillomavirus infection in males with easy collection, increasing positive rate of male HPV infection, and adapting to the new method. T three sorts of samples were collected at same time. Except sequenced and compared of sequences, we also proved the amplified products with 1.5% agarose gel electrophoresis. Through detection and comparison among three sorts of samples, we can make certain the urethral sample, which easily collected, convenient and more sensitive.
Thirdly, clinical applying
Utilizing the established new method, we detected and analyzed prevalence and distribution of human papillomavirus infection in STD males of Shenzhen’s Family Plan Centre. Sample sort was to adopt male urethral secretion.
We drew the conclusion as follows:
1. The established new method, which detect male HPV Subtypes infection, is sensitive, special, convenient, handle easy, and normal control contrast easily set up, et al. The new method is suitable to be used to male clinical diagnosis, epidemical and disease reasons investigation.
2. Nine sorts HPV subtypes are check-up in the test. The sensitivity of membrane special degree of DNA chip can make ten copies DNA molecules.
3. Male HPV infection positive rate was 22.3% in Shenzhen. The most common genotypes were HPV-6, 16 and 73. Ages of Males seeing doctors were from 31 years old to 40 years old, next ones were 21-30 years old, but age wasn’t relative with HPV infection.
4. In multiple infections, 33/73 of double infections were the most. Meanwhile, mixed infections with high-risk genotypes and low-risk genotypes were most prevalent in all co-infections.
5. The relatively right specimen adapted to clinical detecting HPV subtypes is urethral sample. Moreover, the sorting of sample is easily performed.
通过计算机辅助,参考经典通用引物(MY09/11)和加以改进的PGMY09/11设计23种HPV基因亚型的PCR扩增引物,根据Genbank中的HPV的型特异性序列设计及合成探针,制备可同时对18种高危型:HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 83, MM4和5种低危型:6, 11, 42, 43, 44进行分型检测的特定纸质膜芯片。
通过PCR反应,经过PCR产物与HPV膜条(探针条)杂交、显色后,HPV膜条的PC点显示蓝点才是有效膜条,根据其他蓝点显示位置判读HPV基因亚型(单一感染和多重感染)。对膜杂交结果显示单一亚型感染的阳性PCR产物纯化后进行测序。
各型HPV检测段DNA的克隆质粒进行pGEM-T载体构建。重组质粒通过T7启动子引物测序,并与该亚型HPV基因组序列进行比对,以确定构建质粒中所含的各型HPV片段与检测序列完全相同。
将经过工程菌扩增的重组质粒提纯后,应用紫外分光光度计测定核酸浓度,用Tris-HCl配制成106拷贝/ml的原始标准品,再将原始标准品稀释成不同浓度的HPV标准品,通过PCR方法、经杂交、显色后,以确定HPV分型的灵敏度。同时,检测对比男性尿道分泌物、精液及尿液三种类型标本。
得出以下结论:①本课题建立的男性人乳头瘤病毒(HPV)基因亚型检测方法具有灵敏度高、特异性好、实验程序简便、采样方便、易于设定正常对照等优点,适用于临床进行男性HPV感染的诊断,为开展相应的流行病、病因学调查提供切实可行的检测手段。②检测出的男性HPV基因亚型有:HPV-6,11,16,18,33,35,43,56和73型九种。灵敏度可达10个拷贝HPV DNA分子。③临床应用中发现深圳男性HPV检测阳性率是22.3%,男性HPV感染最常见的基因亚型是6、16和73型。就诊的男性中以31岁～40岁的年龄组最多,其次是21岁～30岁年龄组,但发现深圳男性HPV的感染与年龄无相关性。④多重HPV感染中,以双重感染33/73型感染最多,高危型和低危型混合感染最常见。⑤确定临床常规检测男性HPV感染采用尿道分泌物做为采集部位和标本具有较高的敏感性,并且采样相对简便、易行。
本研究主要创新:
同时可以检测23种HPV基因亚型是本研究的创新点,HPV基因芯片法使得临床对HPV亚型检测达到微型化、集约化和标准化,从而保证了诊断的高效、廉价、快速和简便。
Human papillomavirus (HPV)is a sexually transmitted disease widely. Over 100 different HPV types have been identified based on DNA sequence analysis, HPV can be divided into“high-risk”and“low-risk”groups on the basis of their association with oncogenic nature, approximately 60 HPV genotypes are known to infect the genital and urethral tract epithelial sites, at least 13 of which are considered“high-risk”and oncogenic. HPV are considered the important etiologic agents of cervical cancer, which have been researched much many. But they are less relatively the studies to male human papillomavirus infection than the women’s. On one hand, because the sites of physiological anatomy are different, specimens acquisition of males are very difficult than women’s, collecting shedding cells roughly. On the other hand, oncogenic genotypes of HPV to both men and women are varying degree, which in women mean bad results such as cervical cancer, et al. But detection of males infection genotypes may be vital parameter to their transforms, moreover, which are more significant to supervise fatalness of males sexual partners. Infecting HPV males are considered increasing danger to their partners, who are high-risk means of delivery. Several studies have provided estimates of HPV prevalence among men, however, these estimates range widely from 1.0% to 82.9%.Significant barriers to the synthesis and interpretation of these studies are the inconsistency of sampling techniques used, the variety and combinations of sites and specimens sampled, and the testing for different HPV genotypes, for example, the urethra, glans /corona, or scrotum samples, et al. In order to understand the epidemical condition and changing trend, there is urgent to develop an experimentation to adapt to clinical males HPV test, sampling convenient, and be able to detect high-risk and low-risk genotypes simultaneously, so that patients of infecting HPV males can be treated pertinently and epidemical surveillance.
Firstly, establishing methods
Probes of 18 high-risk subtypes and 5 low-risk subtypes were designed according to membrane DNA chip technique and Genbank data about HPV, through hybridizing, et al, 23 males HPV genotype can be detected simultaneously. Through computer, we referred to classically common primers (MY09/11) and improved them to be PGMY09/11 (5’-biotin-CGT CCM ARR GGA WAC TGA TC-3’; 5’-biotin-GCM CAG GGW CAT AAY AAT GG-3’; M=A/C,W=A/T,Y=C/T,R=A/G)as universal primers, andβ- globulin primer PC03/ GH20(5’-biotin-ACA CAA CTG TGT TCA CTA GC-3’; 5’-biotin-GAA GAG CCA AGG ACA GGT AC-3’)as majordomo genes. All primers were signed by biotins about 5’end. We designed the genotyping probes according to Genbank data associated HPV subtypes sequences, and designed the control probe according to Genbank data associatedβ- globulin sequences, all probes were signed by amino acid about 5’end. Analysis probes were fixed on special membrane to make DNA chip according order of number. Type of specimen was male urethral sample with sterile cotton swabs.HPV membrane DNA chip includes 18 high-risk subtypes: HPV-16, 18, 31, 33,35, 39, 45, 51,52, 53, 56, 58, 59, 66, 68, 73, 83, and MM4, and 5 low-risk subtypes: 6, 11, 42, 43 and 44. Through PCR reaction, and After products of PCR reaction hybridized with HPV membrane stripes (probes stripes) and show color, strips were valid when only PC blots show blue dots, meanwhile, HPV subtypes were judged with other sites blue dots on the same strips (single infection and multiple infection). Single subtype HPV infection of position PCR products was sequenced. Cloned plasmid of every HPV subtype DNA was constructed to pGEM-T carrier. Reformed plasmid was sequenced by T7 opener primer and compared with same HPV subtype genome sequence. So can confirm to the detected sequence was fully same as segment with constructed plasmid DNA. Constructed plasmid DNA after purified and amplified with engineering germs was detected nucleic acid concentration by ultraviolet analysis instrument. Original standard sample was diluted to 106 copies with Tris-HCl, which was diluted to serial HPV standard dilutions in different concentration. Sensitivity of analytic HPV genotyping can be confirmed after PCR reaction, hybridization and color display.
On account of shell protein variation of different HPV subtype, there were no cross protecting antibody among HPV subtypes basically, which can make time after time infections between high-risk and high-risk HPV subtypes, or within high-risk and high-risk. We designed primers referring to classically common primers (MY09/11) and improved them to be PGMY09/11 through computer, whose sensitivity was higher than standard MY09/11 primer. In order to avoid amplification failure for HPV primer variation in combined section, we synthesized primer with distinct alkali bases in relatively easy mutation places. Meanwhile, in order to conveniently detect amplified products with reverse hybridiza- tion, we marked one end of the improved primer with biotin and designed specific genotyping probes in HPV 23 subtypes vary section.
Experimental results demonstrate that speciality of genotyping probes can obtain distinguishing rate for one alkaline base, while we make the probes distinguishing over 30% within alkaline bases, so it is highly special the results of hybridization. Moreover, we didn’t found non-special hybridization during sequences analysis of positive samples. The sensitivity of membrane special degree of DNA chip can make ten copies DNA molecules after standard products with 23 HPV subtypes of plasmid construction were detected and show concrete subtype. HPV-6,11,16,18,33,35,43,56 and 73 were detected out , which can prove those above. The new method is suitable to be used to male clinical diagnosis, epidemical and disease reasons investigation.
Secondly, choicing adapt sample sort
Utilizing established new method, we compared three sorts of samples, HPV 23 subtypes were detected in males with comparison of urethral, seminal and urinary specimens. Our aim was to develop a right sorting of specimen for diagnosis of human papillomavirus infection in males with easy collection, increasing positive rate of male HPV infection, and adapting to the new method. T three sorts of samples were collected at same time. Except sequenced and compared of sequences, we also proved the amplified products with 1.5% agarose gel electrophoresis. Through detection and comparison among three sorts of samples, we can make certain the urethral sample, which easily collected, convenient and more sensitive.
Thirdly, clinical applying
Utilizing the established new method, we detected and analyzed prevalence and distribution of human papillomavirus infection in STD males of Shenzhen’s Family Plan Centre. Sample sort was to adopt male urethral secretion.
We drew the conclusion as follows:
1. The established new method, which detect male HPV Subtypes infection, is sensitive, special, convenient, handle easy, and normal control contrast easily set up, et al. The new method is suitable to be used to male clinical diagnosis, epidemical and disease reasons investigation.
2. Nine sorts HPV subtypes are check-up in the test. The sensitivity of membrane special degree of DNA chip can make ten copies DNA molecules.
3. Male HPV infection positive rate was 22.3% in Shenzhen. The most common genotypes were HPV-6, 16 and 73. Ages of Males seeing doctors were from 31 years old to 40 years old, next ones were 21-30 years old, but age wasn’t relative with HPV infection.
4. In multiple infections, 33/73 of double infections were the most. Meanwhile, mixed infections with high-risk genotypes and low-risk genotypes were most prevalent in all co-infections.
5. The relatively right specimen adapted to clinical detecting HPV subtypes is urethral sample. Moreover, the sorting of sample is easily performed.
引文
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