蚊媒病原体综合检测方法的建立及应用
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摘要
蚊媒病是一组以蚊虫为媒介而传播的疾病,包括疟疾、淋巴丝虫病、黄热病、登革热、流行性乙型脑炎、西尼罗热等,是重要的公共健康问题。随着全球气候逐渐上升、城市化进程的加快、旅游和贸易的快速发展、生态环境的不断变化,全球蚊媒传染病发病呈上升趋势,原有疾病的流行区域不断扩展、疾病的流行频度不断增加。
随着疾病谱发生变化,相应的检测和监测技术要及时跟进,以适应新的疾病监测需求。检测及监测蚊虫携带的病原体,在传染病监测中属于早期监测和发现传染病流行的方法,对于蚊媒传播疾病的有效预警和控制有重要的意义。
以往的研究都是针对一种病原体检测,且有些检测方法灵敏度不够高,不能够早期发现蚊媒传染病的流行;此外,从生物学分类角度来看,蚊媒病病原体涉及微生物学与寄生虫学专业,目前蚊媒病监测也是针对单病种,不同病原体分类专业人员分别从自身专业领域进行检测,浪费人力、财力,且各专业独力实施,不利于高效、综合采取防治措施。因而,对于蚊媒病的防治应当综合考虑。
本研究设计对野外采集的蚊虫综合检测多种携带的病原体,如黄病毒属病毒、疟原虫和丝虫。将野外采集蚊虫样本同批次处理后高通量检测,经济、高效检测蚊传病原体,其研究结果可为卫生防病部门提供简化的检测、监测步骤和程序,提高效率和减少重复工作量。
以黄病毒属病毒保守性最高的非结构蛋白5(nonstructural protein,NS5)基因为靶基因,根据GenBank中已报道30多种蚊媒黄病毒属病毒日本脑炎病毒组(Japanese Encephalitis Virus Complex),包括日本(乙型)脑炎病毒(Japanese encephalitis virus, JEV)、登革病毒(Dengue virus, DenV)、黄热病毒(yellow fever virus, YFV)、西尼罗病毒(West Nile virus, WNV)等病毒NS5基因序列,用DNAStar软件设计出3条半巢式PCR通用引物,以JEV cDNA﹑DenV I～IV型cDNA为模板优化反应条件,建立反转录半巢式PCR(RT-hemi-nested PCR)方法;以日本脑炎病毒减毒活疫苗(SA14-14-2株)测定该方法的敏感度,结果显示,RT-hemi-nested PCR方法的最低病毒检出浓度为5.0×10~(-4)PFU/m1,灵敏度与同类方法相比高出一个数量级。
根据GenBank中疟原虫(包括恶性疟原虫﹑间日疟原虫﹑三日疟原虫﹑卵形疟原虫﹑诺氏疟原虫等)SSUrRNA基因和丝虫(马来丝虫,犬恶丝虫等)线粒体基因保守区的序列,用DNAStar软件设计6条半巢式多重PCR通用引物。以恶性疟原虫﹑间日疟原虫﹑马来丝虫DNA为模板优化反应条件,建立半巢式多重PCR方法,然后以恶性疟原虫DNA﹑马来丝虫DNA测定该方法的敏感度。并用该方法检测实验室感染约氏疟原虫的大劣按蚊。所建立的半巢式多重PCR方法检测疟原虫下限为0.53个恶性疟原虫,丝虫检测下限为0.01条马来丝虫。该方法可以明确地检测出大劣按蚊中感染的约氏疟原虫子孢子。
选择云南普洱周边地区作为蚊媒观察点采集蚊虫。按照采集时间将蚊虫每10只分为一个混合样本组,样本少时则每组少于10只。用上述建立的蚊媒黄病毒属RT- hemi-nested PCR检测了54组三带喙库蚊虫样本,其中14组为阳性,经测序确认9组为乙型脑炎病毒﹑3组为登革热II型病毒﹑1组为登革热I型病毒﹑1组未知病毒(与Quang Binh virus相似度最高,为82%)。上述病毒的最小感染率分别为16.7‰,5.55‰,1.85‰和1.85‰。用半巢式多重PCR检测了148组按蚊和三带喙库蚊样本检测疟原虫和丝虫,未发现阳性扩增。
本研究针对蚊媒病防治中的关键需求,建立了一套黄病毒属病毒、疟原虫、丝虫简单、有效、高通量的检测方法,该方法可用于大批量的检测野外采集的蚊虫。经现场蚊虫检测,在云南普洱周边采集的蚊虫体内发现日本脑炎病毒﹑登革病毒I型和II型和未知病毒(属于黄病毒属)。说明在当地人群或动物体内,存在这些黄病毒属日本脑炎组病毒的低度流行。该研究结果应当引起疾病控制有关部门的重视,并对这些地区以及其他类似地区进行进一步的监测;同时本实验建立的检测方法还可用于病人蚊媒病的检测。高通量的蚊虫检测还能够提供可定性和定量的蚊媒携带病原体资料,也可为蚊媒病监测以及可能传入我国的新型蚊媒病病原体的检测以及预警提供了基础资料;随着地理信息系统在蚊媒病监测、预警中的应用,该方法无疑可为蚊媒病的监测提供更有效的资料,使蚊媒病的监测和预警更为准确、更具有科学性。
Mosquito-borne diseases are transmitted by mosquitoes, which include malaria, lymphatic filariasis, yellow fever, Dengue fever, Japanese encephalitis, West Nile fever. etc. The epidemics of mosquito-borne diseases could raise important public health problem. Owing to the global climate warming, accelerating of urbanization, rapid development of tourism and international trading, and the deterioration of enviromnent, the incidence of mosquito-borne disease rises and the affected regions expand.
With the changing pattern of the vector-borne diseases, it is pressed that much sensitive, specific and high throughput detecting methods been developed to fit the need of Disease Surveillance . Detecting of pathogens-carring mosquito are an earlier detection and surveillance approach in forecasting the epidemics of vector-borne diseases, which is crucial for early warning and effective controlling of mosquito-borne diseases. Most studies in surveillance of mosquito-borne diseases are targeted to detect single specific pathogen, and some of methods are not satisfactory due to a lower sensitivity. It is imperative to develop multiple-pathogens detecting methods in surveillance the mosquito-borne diseases.
This study is designed to develop high throughput, multiplex detection hemi-nested PCR methods for screening the field caught mosquitoes. The method includes divide the mosquitoes in pools, and processed the samples in two paralleled vials. One was retro-transcripted for detecting flavivirus. The other one was used for PCR amplifying to detect Plasmodium spp. and filarial worms.
For detecting the flavivirus, the nonstructural proteins gene, NS5 gene was selected as the amplification target. The primers were designed in reference of more than 30 viruses in Japanese Encephalitis Virus Complex found in GenBank , including Japanese encephalitis virus(JEV), Dengue virus( DenV), Yellow fever virus,( YFV), West Nile viruss (WNV), etc. which are endemic in China, or is regarded as the potential imported pathogens.
To optimize the reaction conditions, we used cDNA of JEV, DenV I～IV types as PCR templates. The RT-hemi-nested PCR method was established for the detection of Japanese encephalitis virus Complex. The sensitivity of the method was then further evaluated with attenuated vaccine strain of Japanese encephalitis virus (SA14-14-2 strains). The threshold concentration for detecting the virus was 5.0×10~(-4) PFU/ml, ten-fold sensitive then the similar methods.
A multiplex hemi-nested PCR method was established to amplify the SSU rRNA gene of Plasmodium spp.,( including P. falciparum, P. vivax, P. malaria, P. knowlesi, etc.), and the mitochondria genes sequence of lymphatic filarial worms reported in GenBank (including Brugia malayi, Dirofilariasis immitis, etc.). We designed 6 universal primers for multiplex hemi-nested PCR. And the genomic DNA from P. falciparum, P. vivax, B. malayi were used for optimizing the reaction conditions. The method was evaluated by detecting P. yoelii sporozoites containing Anopheles dirus. The sensitivity of the method was also evaluated with the genomic DNA from P. falciparum and B. malayi. The minimum amout of DNA from P. falciparum that can be detected by the method was equivalent to be 0.53 parasite. And for B. malayi, the amount was equivalent to be 0.01 parasite. The method could clearly detect P. yoelii in An. dirus.
The two methods we established were further evaluated in detection field-collected mosquites. The mosquitoes were trapped from several collecting spots surrounding the Puer cities of Yunnan province, All samples were species identified and allocated into pools, Each mosquito pool contained 10 or less mosquitoes depends on the numbers of each sampling. We used the RT-hemi-nested PCR method to detect 54 pools of Culex tritaeniorhynchus. 14 mosquito pools had positive amplification. Sequencing of the amplicons revealed 9 pools of JEV, 3 pools of DenV type II, 1 pool of DenV type I, and 1 pool of unknown virus (the closest match was Quang Binh virus with 82% similarity). The minimum infection rates for the above viruses are 16.7‰, 5.55‰, 1.85‰, 1.85‰, respectively. 148 pools of mosquitoes was detected for Plasmodium spp., and filarial worms, no positive amplification was found.
The two hemi-nested PCR methods we established are well meeting the need in surveillance of mosquito-borne diseases. The methods show superior in detecting mosquito-borne pathogens simultaneously and high throughputly. Monitoring pathogens-infected mosquitoes is an earlier detection and surveillance approach in forecast the transmitting potential of mosquito-borne diseases. The methods are simple, effective, relative rapid for detection, and is superior over virus culture method in its high-throughput detection. On detecting the field caught mosquitoes, we found JEV, DenV type II and I, unknown virus in mosquitoes collected from Puer, Yunnan province. Health workers in each CDC level should pay more attention to the results, and proceed further surveillance in this region and other similar regions. Meanwhile, the data obtained with the utilizing the methods could also provide experimental documents for mosquitoes surveillance, for diagnosis and monitoring of emerging imported mosquitoes-borne diseases. With the incorporation of the GIS in surveillance and forecasting of mosquito-borne diseases, it is no doubts that the methods we established could provide more reasonable and more effective data.
随着疾病谱发生变化,相应的检测和监测技术要及时跟进,以适应新的疾病监测需求。检测及监测蚊虫携带的病原体,在传染病监测中属于早期监测和发现传染病流行的方法,对于蚊媒传播疾病的有效预警和控制有重要的意义。
以往的研究都是针对一种病原体检测,且有些检测方法灵敏度不够高,不能够早期发现蚊媒传染病的流行;此外,从生物学分类角度来看,蚊媒病病原体涉及微生物学与寄生虫学专业,目前蚊媒病监测也是针对单病种,不同病原体分类专业人员分别从自身专业领域进行检测,浪费人力、财力,且各专业独力实施,不利于高效、综合采取防治措施。因而,对于蚊媒病的防治应当综合考虑。
本研究设计对野外采集的蚊虫综合检测多种携带的病原体,如黄病毒属病毒、疟原虫和丝虫。将野外采集蚊虫样本同批次处理后高通量检测,经济、高效检测蚊传病原体,其研究结果可为卫生防病部门提供简化的检测、监测步骤和程序,提高效率和减少重复工作量。
以黄病毒属病毒保守性最高的非结构蛋白5(nonstructural protein,NS5)基因为靶基因,根据GenBank中已报道30多种蚊媒黄病毒属病毒日本脑炎病毒组(Japanese Encephalitis Virus Complex),包括日本(乙型)脑炎病毒(Japanese encephalitis virus, JEV)、登革病毒(Dengue virus, DenV)、黄热病毒(yellow fever virus, YFV)、西尼罗病毒(West Nile virus, WNV)等病毒NS5基因序列,用DNAStar软件设计出3条半巢式PCR通用引物,以JEV cDNA﹑DenV I～IV型cDNA为模板优化反应条件,建立反转录半巢式PCR(RT-hemi-nested PCR)方法;以日本脑炎病毒减毒活疫苗(SA14-14-2株)测定该方法的敏感度,结果显示,RT-hemi-nested PCR方法的最低病毒检出浓度为5.0×10~(-4)PFU/m1,灵敏度与同类方法相比高出一个数量级。
根据GenBank中疟原虫(包括恶性疟原虫﹑间日疟原虫﹑三日疟原虫﹑卵形疟原虫﹑诺氏疟原虫等)SSUrRNA基因和丝虫(马来丝虫,犬恶丝虫等)线粒体基因保守区的序列,用DNAStar软件设计6条半巢式多重PCR通用引物。以恶性疟原虫﹑间日疟原虫﹑马来丝虫DNA为模板优化反应条件,建立半巢式多重PCR方法,然后以恶性疟原虫DNA﹑马来丝虫DNA测定该方法的敏感度。并用该方法检测实验室感染约氏疟原虫的大劣按蚊。所建立的半巢式多重PCR方法检测疟原虫下限为0.53个恶性疟原虫,丝虫检测下限为0.01条马来丝虫。该方法可以明确地检测出大劣按蚊中感染的约氏疟原虫子孢子。
选择云南普洱周边地区作为蚊媒观察点采集蚊虫。按照采集时间将蚊虫每10只分为一个混合样本组,样本少时则每组少于10只。用上述建立的蚊媒黄病毒属RT- hemi-nested PCR检测了54组三带喙库蚊虫样本,其中14组为阳性,经测序确认9组为乙型脑炎病毒﹑3组为登革热II型病毒﹑1组为登革热I型病毒﹑1组未知病毒(与Quang Binh virus相似度最高,为82%)。上述病毒的最小感染率分别为16.7‰,5.55‰,1.85‰和1.85‰。用半巢式多重PCR检测了148组按蚊和三带喙库蚊样本检测疟原虫和丝虫,未发现阳性扩增。
本研究针对蚊媒病防治中的关键需求,建立了一套黄病毒属病毒、疟原虫、丝虫简单、有效、高通量的检测方法,该方法可用于大批量的检测野外采集的蚊虫。经现场蚊虫检测,在云南普洱周边采集的蚊虫体内发现日本脑炎病毒﹑登革病毒I型和II型和未知病毒(属于黄病毒属)。说明在当地人群或动物体内,存在这些黄病毒属日本脑炎组病毒的低度流行。该研究结果应当引起疾病控制有关部门的重视,并对这些地区以及其他类似地区进行进一步的监测;同时本实验建立的检测方法还可用于病人蚊媒病的检测。高通量的蚊虫检测还能够提供可定性和定量的蚊媒携带病原体资料,也可为蚊媒病监测以及可能传入我国的新型蚊媒病病原体的检测以及预警提供了基础资料;随着地理信息系统在蚊媒病监测、预警中的应用,该方法无疑可为蚊媒病的监测提供更有效的资料,使蚊媒病的监测和预警更为准确、更具有科学性。
Mosquito-borne diseases are transmitted by mosquitoes, which include malaria, lymphatic filariasis, yellow fever, Dengue fever, Japanese encephalitis, West Nile fever. etc. The epidemics of mosquito-borne diseases could raise important public health problem. Owing to the global climate warming, accelerating of urbanization, rapid development of tourism and international trading, and the deterioration of enviromnent, the incidence of mosquito-borne disease rises and the affected regions expand.
With the changing pattern of the vector-borne diseases, it is pressed that much sensitive, specific and high throughput detecting methods been developed to fit the need of Disease Surveillance . Detecting of pathogens-carring mosquito are an earlier detection and surveillance approach in forecasting the epidemics of vector-borne diseases, which is crucial for early warning and effective controlling of mosquito-borne diseases. Most studies in surveillance of mosquito-borne diseases are targeted to detect single specific pathogen, and some of methods are not satisfactory due to a lower sensitivity. It is imperative to develop multiple-pathogens detecting methods in surveillance the mosquito-borne diseases.
This study is designed to develop high throughput, multiplex detection hemi-nested PCR methods for screening the field caught mosquitoes. The method includes divide the mosquitoes in pools, and processed the samples in two paralleled vials. One was retro-transcripted for detecting flavivirus. The other one was used for PCR amplifying to detect Plasmodium spp. and filarial worms.
For detecting the flavivirus, the nonstructural proteins gene, NS5 gene was selected as the amplification target. The primers were designed in reference of more than 30 viruses in Japanese Encephalitis Virus Complex found in GenBank , including Japanese encephalitis virus(JEV), Dengue virus( DenV), Yellow fever virus,( YFV), West Nile viruss (WNV), etc. which are endemic in China, or is regarded as the potential imported pathogens.
To optimize the reaction conditions, we used cDNA of JEV, DenV I～IV types as PCR templates. The RT-hemi-nested PCR method was established for the detection of Japanese encephalitis virus Complex. The sensitivity of the method was then further evaluated with attenuated vaccine strain of Japanese encephalitis virus (SA14-14-2 strains). The threshold concentration for detecting the virus was 5.0×10~(-4) PFU/ml, ten-fold sensitive then the similar methods.
A multiplex hemi-nested PCR method was established to amplify the SSU rRNA gene of Plasmodium spp.,( including P. falciparum, P. vivax, P. malaria, P. knowlesi, etc.), and the mitochondria genes sequence of lymphatic filarial worms reported in GenBank (including Brugia malayi, Dirofilariasis immitis, etc.). We designed 6 universal primers for multiplex hemi-nested PCR. And the genomic DNA from P. falciparum, P. vivax, B. malayi were used for optimizing the reaction conditions. The method was evaluated by detecting P. yoelii sporozoites containing Anopheles dirus. The sensitivity of the method was also evaluated with the genomic DNA from P. falciparum and B. malayi. The minimum amout of DNA from P. falciparum that can be detected by the method was equivalent to be 0.53 parasite. And for B. malayi, the amount was equivalent to be 0.01 parasite. The method could clearly detect P. yoelii in An. dirus.
The two methods we established were further evaluated in detection field-collected mosquites. The mosquitoes were trapped from several collecting spots surrounding the Puer cities of Yunnan province, All samples were species identified and allocated into pools, Each mosquito pool contained 10 or less mosquitoes depends on the numbers of each sampling. We used the RT-hemi-nested PCR method to detect 54 pools of Culex tritaeniorhynchus. 14 mosquito pools had positive amplification. Sequencing of the amplicons revealed 9 pools of JEV, 3 pools of DenV type II, 1 pool of DenV type I, and 1 pool of unknown virus (the closest match was Quang Binh virus with 82% similarity). The minimum infection rates for the above viruses are 16.7‰, 5.55‰, 1.85‰, 1.85‰, respectively. 148 pools of mosquitoes was detected for Plasmodium spp., and filarial worms, no positive amplification was found.
The two hemi-nested PCR methods we established are well meeting the need in surveillance of mosquito-borne diseases. The methods show superior in detecting mosquito-borne pathogens simultaneously and high throughputly. Monitoring pathogens-infected mosquitoes is an earlier detection and surveillance approach in forecast the transmitting potential of mosquito-borne diseases. The methods are simple, effective, relative rapid for detection, and is superior over virus culture method in its high-throughput detection. On detecting the field caught mosquitoes, we found JEV, DenV type II and I, unknown virus in mosquitoes collected from Puer, Yunnan province. Health workers in each CDC level should pay more attention to the results, and proceed further surveillance in this region and other similar regions. Meanwhile, the data obtained with the utilizing the methods could also provide experimental documents for mosquitoes surveillance, for diagnosis and monitoring of emerging imported mosquitoes-borne diseases. With the incorporation of the GIS in surveillance and forecasting of mosquito-borne diseases, it is no doubts that the methods we established could provide more reasonable and more effective data.
引文
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