实时荧光定量RT-PCR检测1型鸭病毒性肝炎方法的建立和应用
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摘要
本文围绕检测1型鸭病毒性肝炎病毒(DHV-1)的一步法荧光定量反转录聚合酶链式反应(real-time reverse transcriptase-polymerase chain reaction,rRT-PCR)诊断方法的建立和应用开展了系列研究:①建立了基于DHV-1 3D基因的一步法rRT-PCR;②应用该方法对DHV-1强毒在鸭胚内的分布规律和弱毒在鸡胚内的分布规律进行了研究;③定量检测人工感染DHV-1的雏鸭体内病毒动态分布情况;④定量检测感染DHV-1强毒后耐过雏鸭的病毒排泄规律,并对四川部分鸭场的健康鸭群和养鸭环境进行了流行病学调查。得到如下结果:
根据DHV-1 3D区域序列,设计并合成了一对能特异性扩增87bp片段的引物和TaqMan~(TM)探针,建立了一步法rRT-PCR诊断方法。该法在DHV-1 RNA含量为1×10~(10)-1×10~5copies有较好的线性关系;该法建立的标准曲线为Y=-3.431X+46.443,相关系数为1.000,PCR循环效率为95.6%,检测DHV-1 RNA模板的灵敏度为10copies/反应;特异性试验表明只对DHV-1基因组呈阳性反应,而对鸭瘟病毒、番鸭细小病毒、鹅细小病毒、雏鹅新型病毒性肠炎病毒、禽流感病毒(H5N2亚型)、鸭病毒性肿头出血症病毒、鸭源多杀性巴氏杆菌(5:A)、鸭源肠炎沙门氏菌、鸭源鼠伤寒沙门氏菌和鸭源致病性大肠杆菌(078)、正常鸭胚尿囊液和健康鸭肝组织呈阴性反应。用建立的方法检测了DHV-1强毒在鸭胚中的增殖规律以及弱毒在鸡胚中的增殖规律,鸭胚和鸡胚分别在接毒后28-40 h、44-56 h病毒含量达到最大,胚体中为10~(11)copies/g数量级,尿囊液中为10~8-10~(10)copies/ml数量级。死亡胚内尿囊膜、肌肉中的病毒含量高于尿囊液、心、肝、脾、脑、肠。分别应用该技术和传统病毒分离和中和试验法检测10份来自人工感染雏鸭的肝脏以及40份临床疑似病鸭的肝脏,结果显示两种方法对人工感染病料的阳性检测结果符合率为100%,而rRT-PCR对临床疑似病料的阳性检测率极显著(P＜0.01)高于病毒分离和中和试验。研究结果表明,该方法快速、灵敏、特异、重复性好且能实时定量检测,可用于DHV-1感染疑似病例检测、DHV-1体内动态分布规律研究及分子流行病学调查。
应用建立的rRT-PCR方法对经肌注和口服途径人工感染DHV-1雏鸭的组织器官进行检测,结果表明:经肌注接种DHV-1后最早可在1 h内从肝脏和哈德氏腺中检测到,2 h即可在肌肉、心脏、脾脏、肺脏、肾脏、十二指肠、胸腺中检测到;4 h在空肠、回肠、盲肠中检测到阳性结果;感染后6 h所有采集的样品中均能检测到DHV-1核酸的存在。6-72 h之间各组织器官均能检测到DHV-1,肌注组出现高峰期的时间集中在24-48 h。统计显示,肝脏、胸腺、哈德氏腺、盲肠、肾脏和肌肉的阳性拷贝数高于其它部位。除了肌肉、心脏、肺脏、胸腺、哈德氏腺和脑以外的其他组织器官直至攻毒后2 w仍然能检测到病毒。经口服途径接种后,2 h即在肝脏和哈德氏腺中最早被检测到,4 h可在口服组的肌肉、心脏、脾脏、肾脏、十二指肠、胸腺中被检测到;感染后6 h,除了肺脏、胰、盲肠和脑以外,其它所有采集的样品中均能检测到DHV-1核酸的存在。36-120 h之间全部被检组织器官呈阳性反应,口服组出现高峰期的时间集中在48-72 h。肝脏、胸腺、哈德氏腺、直肠和心脏的阳性拷贝数高于其它部位。到攻毒后2 w,除了肌肉、肺脏、哈德氏腺和脑以外的其他组织器官仍然能检测到病毒。
对肌注和口服两种途径感染DHV-1强毒耐过的雏鸭进行粪便排毒的检测,结果表明:口服组在60 d之内已经有2只(2/5)停止排毒,到第120 d已经全部(5/5)停止排毒。而肌注组在90 d之内仅有1只(1/5)停止排毒,到了120 d仍有2只(2/5)在排毒。同居组从第5 d开始检测出有雏鸭感染,至第14 d有4只(4/10)雏鸭感染。对四川部分地区鸭场采集样品进行检测,结果表明:饮水、戏水池、环境拭子和肛门拭子中均有可能检测出DHV-1,其中,从戏水池和肛门拭子中检出DHV-1的阳性率较高,饮水中检出DHV-1的概率较小,说明四川地区的鸭场有受到DHV-1威胁的可能,除了鸭的体内环境有利于病毒的生长繁殖外,非流动的戏水池环境也可存储病毒。
The dissertation revolves around the development and application of a one-step real-time TaqMan RT-PCR assay to detect duck hepatitis virus type 1(DHV-1):①Development of a one-step real-time TaqMan RT-PCR assay based on the 3D gene of DHV-1 genome;②Using the new assay,the distribution and amount of virulent strain in duck embryos were detected as well as the attenuated vaccine strain in chicken embryos;③Detection of DHV-1 in the tissues of artificially infected ducklings;④Detection of DHV-1 in the stool of tolerated ducklings which were artificially infected with virulent strain and a molecular epidemiology survey of DHV-1 in ducks and conditions of Sichuan Province.The results are as follows:
A one-step real-time reverse-transcription polymerase chain reaction assay(rRT-PCR) was developed for efficient detection of DHV-1.A pair of specific primers which showed 87bp fragment was designed against the conserved region in the 3D gene with a single conserved TaqMan~(TM) probe.It could obtain excellent linear when the DHV-1 RNA concentration between 1×10~(10) and 1×10~5 copies.The detection limit of this assay was 10 viral genomic copies per reaction and it was highly specific to DHV-1 genera,whereas DPV,MPV,GPV,NGVEV,AIV(H5N2),DSHDV,Duck origin Oasturella multocida(5:A), Duck origin salmonella enteritidis,duck origin salmonella typhimurium,duck origin Escherichia coli(O78),allantoic fluid of normal duck embryos and liver of health duck showed negative results.Then,the rRT-PCR assay was used to determine the distribution and concentration of DHV-1 virulent strain in duck embryos as well as the DHV-1 attenuated vaccine strain in chicken embryos.The results revealed that the copy number of DHV-1 reached a peak in duck embryos and chicken embryos at 28-40 h,44-56 h post inoculation respectively,maintaining 10~(11) copies/g level in embryoid bodies and 10~8 -10~(10) copies/ml level in allantoic fluid.The RNA copy number in chorioallantoic membrane and muscle was higher than that in allantoic fluid,heart,liver,spleen,brain,intestine from dead duck and chicken embryos inoculated with DHV-1.Using this method and neutralization test to detect 10 liver samples taking from ducklings after artificially infected and 40 clinical liver samples taking from suspected ducklings showed that the positive results of artificially infected samples were the same,while the rRT-PCR method was more sensitive than neutralization test for clinical samples detection,and the positive rate was significant difference(P＜0.01).In conclusion,the rRT-PCR assay is rapid, sensitive,specific,reliable and quantitive.It will be a powerful tool for DHV-1 suspected case detection,distribution pattern of DHV-1 in vivo and molecular epidemiological screening.
We used this rRT-PCR assay to detect DHV-1 of the artificially infected ducklings and the results showed that:after inoculated intramuscularly,the liver and Harder's glands were positive at 1 h post inoculation;and the muscle,heart,spleen,lung,kidney, duodenum and thymus were positive at 2 h.The positive results of jejunum,ileum and cecum came out at 4 h,and all the samples were positive at 6 h.All the tissues were positive at 6-72 h and the copy number of DHV-1 RNA in each tissue reached a peak at 24-48 h post inoculation,with the liver,thymus,Harder's glands,cecum,kidney and muscle containing high concentrations of DHV-1.All the samples were still positive until 2 w post inoculation except muscle,heart,lung,thymus,Harder's glands and brain.After inoculated orally,the liver and Harder's glands were positive at 2 h post inoculation;and the muscle,heart,spleen,kidney,duodenum and thymus were positive at 4 h.All the samples were positive at 6 h except lung,pancreas,cecum and brain.All the tissues were positive at 36-120 h and the copy number of DHV-1 RNA in each tissue reached a peak at 48-72 h post inoculation,with the liver,thymus,Harder's glands,rectum and heart containing high concentrations of DHV-1.All the samples were still positive until 2 w post inoculation except muscle,lung,Harder's glands and brain.
We detected DHV-1 in the stool of tolerated ducklings which were artificially infected with virulent strain by oral and intramuscular challenge,and the results showed virus excretion pattern:in the oral challenge group,there are 2 of 5 ducks detected negative with stool within 60 days post inoculation,and all the stool of 5 ducks were negative at 120 d post inoculation.However,in the intramuscular challenge group,there was only 1 of 5 ducks detected negative with stool within 90 days post inoculation,and 2 of 5 ducks were still positive at 120 d post inoculation.1 of 10 cohabited ducks was started to be detected positive at 5 d post cohabitation,and there were 4 ducks detected positive until 14 d.The detection of samples collected from duck rising farms in Sichuan Province showed that, DHV-1 could be detected in the drinking water,natatorium,condition swab and anus swab with natatorium and anus swab containing high concentrations of DHV-1,drinking water containing low concentrations of it.The duck rising farms in Sichuan Province could be under the threat of DHV-1 infection,with the virus growth in vivo of ducks as well as in the illiquid natatorium.
根据DHV-1 3D区域序列,设计并合成了一对能特异性扩增87bp片段的引物和TaqMan~(TM)探针,建立了一步法rRT-PCR诊断方法。该法在DHV-1 RNA含量为1×10~(10)-1×10~5copies有较好的线性关系;该法建立的标准曲线为Y=-3.431X+46.443,相关系数为1.000,PCR循环效率为95.6%,检测DHV-1 RNA模板的灵敏度为10copies/反应;特异性试验表明只对DHV-1基因组呈阳性反应,而对鸭瘟病毒、番鸭细小病毒、鹅细小病毒、雏鹅新型病毒性肠炎病毒、禽流感病毒(H5N2亚型)、鸭病毒性肿头出血症病毒、鸭源多杀性巴氏杆菌(5:A)、鸭源肠炎沙门氏菌、鸭源鼠伤寒沙门氏菌和鸭源致病性大肠杆菌(078)、正常鸭胚尿囊液和健康鸭肝组织呈阴性反应。用建立的方法检测了DHV-1强毒在鸭胚中的增殖规律以及弱毒在鸡胚中的增殖规律,鸭胚和鸡胚分别在接毒后28-40 h、44-56 h病毒含量达到最大,胚体中为10~(11)copies/g数量级,尿囊液中为10~8-10~(10)copies/ml数量级。死亡胚内尿囊膜、肌肉中的病毒含量高于尿囊液、心、肝、脾、脑、肠。分别应用该技术和传统病毒分离和中和试验法检测10份来自人工感染雏鸭的肝脏以及40份临床疑似病鸭的肝脏,结果显示两种方法对人工感染病料的阳性检测结果符合率为100%,而rRT-PCR对临床疑似病料的阳性检测率极显著(P＜0.01)高于病毒分离和中和试验。研究结果表明,该方法快速、灵敏、特异、重复性好且能实时定量检测,可用于DHV-1感染疑似病例检测、DHV-1体内动态分布规律研究及分子流行病学调查。
应用建立的rRT-PCR方法对经肌注和口服途径人工感染DHV-1雏鸭的组织器官进行检测,结果表明:经肌注接种DHV-1后最早可在1 h内从肝脏和哈德氏腺中检测到,2 h即可在肌肉、心脏、脾脏、肺脏、肾脏、十二指肠、胸腺中检测到;4 h在空肠、回肠、盲肠中检测到阳性结果;感染后6 h所有采集的样品中均能检测到DHV-1核酸的存在。6-72 h之间各组织器官均能检测到DHV-1,肌注组出现高峰期的时间集中在24-48 h。统计显示,肝脏、胸腺、哈德氏腺、盲肠、肾脏和肌肉的阳性拷贝数高于其它部位。除了肌肉、心脏、肺脏、胸腺、哈德氏腺和脑以外的其他组织器官直至攻毒后2 w仍然能检测到病毒。经口服途径接种后,2 h即在肝脏和哈德氏腺中最早被检测到,4 h可在口服组的肌肉、心脏、脾脏、肾脏、十二指肠、胸腺中被检测到;感染后6 h,除了肺脏、胰、盲肠和脑以外,其它所有采集的样品中均能检测到DHV-1核酸的存在。36-120 h之间全部被检组织器官呈阳性反应,口服组出现高峰期的时间集中在48-72 h。肝脏、胸腺、哈德氏腺、直肠和心脏的阳性拷贝数高于其它部位。到攻毒后2 w,除了肌肉、肺脏、哈德氏腺和脑以外的其他组织器官仍然能检测到病毒。
对肌注和口服两种途径感染DHV-1强毒耐过的雏鸭进行粪便排毒的检测,结果表明:口服组在60 d之内已经有2只(2/5)停止排毒,到第120 d已经全部(5/5)停止排毒。而肌注组在90 d之内仅有1只(1/5)停止排毒,到了120 d仍有2只(2/5)在排毒。同居组从第5 d开始检测出有雏鸭感染,至第14 d有4只(4/10)雏鸭感染。对四川部分地区鸭场采集样品进行检测,结果表明:饮水、戏水池、环境拭子和肛门拭子中均有可能检测出DHV-1,其中,从戏水池和肛门拭子中检出DHV-1的阳性率较高,饮水中检出DHV-1的概率较小,说明四川地区的鸭场有受到DHV-1威胁的可能,除了鸭的体内环境有利于病毒的生长繁殖外,非流动的戏水池环境也可存储病毒。
The dissertation revolves around the development and application of a one-step real-time TaqMan RT-PCR assay to detect duck hepatitis virus type 1(DHV-1):①Development of a one-step real-time TaqMan RT-PCR assay based on the 3D gene of DHV-1 genome;②Using the new assay,the distribution and amount of virulent strain in duck embryos were detected as well as the attenuated vaccine strain in chicken embryos;③Detection of DHV-1 in the tissues of artificially infected ducklings;④Detection of DHV-1 in the stool of tolerated ducklings which were artificially infected with virulent strain and a molecular epidemiology survey of DHV-1 in ducks and conditions of Sichuan Province.The results are as follows:
A one-step real-time reverse-transcription polymerase chain reaction assay(rRT-PCR) was developed for efficient detection of DHV-1.A pair of specific primers which showed 87bp fragment was designed against the conserved region in the 3D gene with a single conserved TaqMan~(TM) probe.It could obtain excellent linear when the DHV-1 RNA concentration between 1×10~(10) and 1×10~5 copies.The detection limit of this assay was 10 viral genomic copies per reaction and it was highly specific to DHV-1 genera,whereas DPV,MPV,GPV,NGVEV,AIV(H5N2),DSHDV,Duck origin Oasturella multocida(5:A), Duck origin salmonella enteritidis,duck origin salmonella typhimurium,duck origin Escherichia coli(O78),allantoic fluid of normal duck embryos and liver of health duck showed negative results.Then,the rRT-PCR assay was used to determine the distribution and concentration of DHV-1 virulent strain in duck embryos as well as the DHV-1 attenuated vaccine strain in chicken embryos.The results revealed that the copy number of DHV-1 reached a peak in duck embryos and chicken embryos at 28-40 h,44-56 h post inoculation respectively,maintaining 10~(11) copies/g level in embryoid bodies and 10~8 -10~(10) copies/ml level in allantoic fluid.The RNA copy number in chorioallantoic membrane and muscle was higher than that in allantoic fluid,heart,liver,spleen,brain,intestine from dead duck and chicken embryos inoculated with DHV-1.Using this method and neutralization test to detect 10 liver samples taking from ducklings after artificially infected and 40 clinical liver samples taking from suspected ducklings showed that the positive results of artificially infected samples were the same,while the rRT-PCR method was more sensitive than neutralization test for clinical samples detection,and the positive rate was significant difference(P＜0.01).In conclusion,the rRT-PCR assay is rapid, sensitive,specific,reliable and quantitive.It will be a powerful tool for DHV-1 suspected case detection,distribution pattern of DHV-1 in vivo and molecular epidemiological screening.
We used this rRT-PCR assay to detect DHV-1 of the artificially infected ducklings and the results showed that:after inoculated intramuscularly,the liver and Harder's glands were positive at 1 h post inoculation;and the muscle,heart,spleen,lung,kidney, duodenum and thymus were positive at 2 h.The positive results of jejunum,ileum and cecum came out at 4 h,and all the samples were positive at 6 h.All the tissues were positive at 6-72 h and the copy number of DHV-1 RNA in each tissue reached a peak at 24-48 h post inoculation,with the liver,thymus,Harder's glands,cecum,kidney and muscle containing high concentrations of DHV-1.All the samples were still positive until 2 w post inoculation except muscle,heart,lung,thymus,Harder's glands and brain.After inoculated orally,the liver and Harder's glands were positive at 2 h post inoculation;and the muscle,heart,spleen,kidney,duodenum and thymus were positive at 4 h.All the samples were positive at 6 h except lung,pancreas,cecum and brain.All the tissues were positive at 36-120 h and the copy number of DHV-1 RNA in each tissue reached a peak at 48-72 h post inoculation,with the liver,thymus,Harder's glands,rectum and heart containing high concentrations of DHV-1.All the samples were still positive until 2 w post inoculation except muscle,lung,Harder's glands and brain.
We detected DHV-1 in the stool of tolerated ducklings which were artificially infected with virulent strain by oral and intramuscular challenge,and the results showed virus excretion pattern:in the oral challenge group,there are 2 of 5 ducks detected negative with stool within 60 days post inoculation,and all the stool of 5 ducks were negative at 120 d post inoculation.However,in the intramuscular challenge group,there was only 1 of 5 ducks detected negative with stool within 90 days post inoculation,and 2 of 5 ducks were still positive at 120 d post inoculation.1 of 10 cohabited ducks was started to be detected positive at 5 d post cohabitation,and there were 4 ducks detected positive until 14 d.The detection of samples collected from duck rising farms in Sichuan Province showed that, DHV-1 could be detected in the drinking water,natatorium,condition swab and anus swab with natatorium and anus swab containing high concentrations of DHV-1,drinking water containing low concentrations of it.The duck rising farms in Sichuan Province could be under the threat of DHV-1 infection,with the virus growth in vivo of ducks as well as in the illiquid natatorium.
引文
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