河北省漢坦病毒宿主動物攜帶病毒特性與種群遺傳特性分析研究
發(fā)布時間：2018-08-12 18:39
【摘要】： 目的:了解河北省腎綜合征出血熱疫源地鼠種構(gòu)成、帶毒率及宿主動物所攜帶漢坦病毒與種群遺傳特性間的關(guān)系,指導(dǎo)對本病的預(yù)防和控制。 方法: 1疫源地調(diào)查,夾夜法捕鼠,無菌取鼠肺; 2用冷凍切片機切鼠肺,冷丙酮固定,間接免疫熒光法篩選漢坦病毒陽性鼠肺; 3從陽性和陰性鼠肺中提取鼠基因組DNA; 4根據(jù)文獻以及GENEBANK中已知的微衛(wèi)星位點查找特異性引物,用聚合酶鏈反應(yīng)(PCR)進行擴增,并進行電泳和觀察結(jié)果; 5根據(jù)電泳結(jié)果顯示,比較同一地區(qū)和不同地區(qū)陰性陽性標本間的差異,并根據(jù)相似系數(shù)計算不同地區(qū)間種群的親緣關(guān)系。 結(jié)果: 1 2008年的鼠間疫情資料顯示褐家鼠占總捕獲鼠種的70.39%,是河北省的優(yōu)勢鼠種。河北省居民區(qū)鼠種主要為褐家鼠和小家鼠,分別占居民區(qū)總捕獲鼠種的73.38%和24.10%;野外鼠種主要為褐家鼠、大倉鼠和小家鼠,分別占野外總捕獲鼠種的54.72%、20.75%和13.21%。河北省居民區(qū)鼠密度為2.16%,高于野外鼠密度(0.53%)。 2將331份鼠肺冷凍切片,冷丙酮固定后用間接免疫熒光法檢測,陽性鼠肺共1份,陽性率為0.30%。提示我省腎綜合征出血熱疫源地鼠帶毒率較低,帶毒鼠為宅區(qū)褐家鼠,野外及其它鼠種未檢出。 3通過對同一地區(qū)攜帶與未攜帶漢坦病毒的褐家鼠間微衛(wèi)星擴增結(jié)果比較得出,D4mit20、D16mit4和D18mit1三個位點均不穩(wěn)定,但D16mit4位點擴增出的條帶在唐山地區(qū)穩(wěn)定存在,D18mit1位點僅可在滄州地區(qū)的陽性標本上擴增出條帶,D2mit12、D3mit15、D3mit22、D7mit26、D12wox10、D12wox11和D14mit2這7個位點上陽性與陰性標本的擴增結(jié)果未顯現(xiàn)出明顯差別。 4通過對同一地區(qū)未攜帶漢坦病毒的褐家鼠間微衛(wèi)星擴增結(jié)果比較得出,D4mit20、D16mit4和D18mit1三個位點極不穩(wěn)定,但偶爾會出現(xiàn),不能作為遺傳特征的標記位點;D2mit12、D7mit26和D12wox10三個位點在各地區(qū)內(nèi)比較穩(wěn)定且發(fā)生變異小;D3mit15、D3mit22、D12wox11和D14mit2四個位點在各地區(qū)內(nèi)遺傳變異大,表現(xiàn)出多態(tài)性,鼠個體間存在遺傳差別。 5通過對不同地區(qū)未攜帶漢坦病毒的褐家鼠間微衛(wèi)星擴增結(jié)果比較得出,褐家鼠在D3mit15、D12wox10、D12wox11和D14mit2位點上存在地區(qū)間差異,表明不同地區(qū)的褐家鼠在遺傳上存在差別,其余微衛(wèi)星位點未顯示出差別。經(jīng)過比較相似系數(shù)發(fā)現(xiàn),邯鄲與保定、石家莊與唐山、滄州與邯鄲的褐家鼠種群間親緣關(guān)系較遠,遺傳距離較大。滄州與唐山的相似系數(shù)為0.8,親緣關(guān)系較近。 6通過對不同地區(qū)攜帶漢坦病毒的褐家鼠間微衛(wèi)星擴增結(jié)果比較得出,D16mit4位點在唐山地區(qū)的標本上擴增出條帶;D18mit1位點在滄州標本上也可以擴增出條帶,但不穩(wěn)定;D2mit12、D3mit22、D12wox10和D14mit2位點上各地區(qū)標本擴增出的條帶電泳位置一致,呈單態(tài)性,這三個地區(qū)間的陽性標本在這些位點上無差異;D7mit26、D12wox11和D3mit15位點在三個地區(qū)擴增結(jié)果穩(wěn)定,但表現(xiàn)出多態(tài)性;D4mit20位點擴增結(jié)果穩(wěn)定,但與預(yù)期結(jié)果不符。經(jīng)過比較相似系數(shù)發(fā)現(xiàn),唐山、滄州和保定三個地區(qū)攜帶有漢坦病毒的褐家鼠在這十個位點上的遺傳距離不大,親緣關(guān)系較近。 結(jié)論: 1褐家鼠、小家鼠是河北省居民區(qū)主要鼠種,褐家鼠、大倉鼠和小家鼠是野外主要鼠種。褐家鼠是河北省的優(yōu)勢鼠種,并且是腎綜合征出血熱的主要傳染源。 2經(jīng)過對攜帶漢坦病毒和未攜帶漢坦病毒的褐家鼠在十個微衛(wèi)星位點的比較,未發(fā)現(xiàn)兩者之間存在明顯差別,還不能說明褐家鼠感染漢坦病毒與其遺傳因素有關(guān)。 3通過計算相似系數(shù)得出,滄州與唐山的褐家鼠親緣關(guān)系較近;邯鄲與保定、邯鄲與滄州、石家莊與唐山間的褐家鼠親緣關(guān)系較遠,遺傳距離大。不同地區(qū)的褐家鼠存在遺傳上的差別。 4在所研究微衛(wèi)星位點中,D16mit4、D18mit1和D4mit20三個位點極不穩(wěn)定;D2mit12、D7mit26和D12wox10位點表現(xiàn)出單態(tài)性,遺傳差異小;D3mit15、D3mit22、D12wox11和D14mit2位點表現(xiàn)出多態(tài)性,遺傳差異大。
[Abstract]:Objective: To understand the species composition, virulence rate and the relationship between Hantavirus carried by host animals and the genetic characteristics of the population in the epidemic focus of hemorrhagic fever with renal syndrome (HFRS) in Hebei Province, and to guide the prevention and control of the disease.
Method:
1 the investigation of the focus of the epidemic.
The lungs of mice were cut by frozen section machine, fixed by cold acetone, and screened by indirect immunofluorescence.
3 extract genomic DNA from the lungs of positive and negative mice.
According to the literature and the known microsatellite sites in GENEBANK, specific primers were searched and amplified by polymerase chain reaction (PCR), and the results of electrophoresis and observation were carried out.
5. The results of electrophoresis showed that the difference between the negative and positive samples in the same region and different regions was compared, and the genetic relationship among the populations in different regions was calculated according to the similarity coefficient.
Result:
The epidemic data in 2008 showed that Rattus norvegicus accounted for 70.39% of the total captured species, and it was the dominant species in Hebei Province. %, 20.75% and 13.21%. of rodent density in Hebei province were 2.16%, higher than that of rodent density (0.53%).
2. 331 frozen sections of rat lungs were fixed with cold acetone and detected by indirect immunofluorescence method. The positive rate was 0.30%. It was suggested that the rodents in the epidemic focus of hemorrhagic fever with renal syndrome in our province had a low virulence rate, and the rodents were Rattus norvegicus in the residential area, but not found in the field and other rodents.
The results of microsatellite amplification showed that D4mit20, D16mit4 and D18mit1 were unstable, but the amplified bands of D16mit4 were stable in Tangshan area. D18mit1 could only be amplified in positive samples of Cangzhou area, D2mit12, D3mit15, D3. There was no significant difference in the amplification results of positive and negative specimens at mit22, D7mit26, D12wox10, D12wox11 and D14mit2 loci.
The results of microsatellite amplification showed that D4mit20, D16mit4 and D18mit1 loci were very unstable, but occasionally appeared and could not be used as marker loci for genetic characteristics; D2mit12, D7mit26 and D12wox10 loci were stable and had little variation in each region; D3mit15, D3mit2 were stable and the mutation was small. 2. The genetic variations of D12wox11 and D14mit2 loci in different regions were large, showing polymorphism, and there were genetic differences among individuals.
5 Comparing the results of microsatellite amplification among Rattus norvegicus without Hantaan virus in different areas, we found that there were regional differences in D3mit15, D12wox10, D12wox11 and D14mit2 loci in Rattus norvegicus, indicating that there were genetic differences among Rattus norvegicus in different areas, and the other microsatellite loci did not show differences. The genetic distance between the populations of Rattus norvegicus in Danzhou and Baoding, Shijiazhuang and Tangshan, Cangzhou and Handan was relatively large, and the similarity coefficient between Cangzhou and Tangshan was 0.8.
6. Comparing the results of microsatellite amplification of Hantavirus in Rattus norvegicus from different regions, we found that the D16mit4 locus amplified a band in Tangshan region, the D18mit1 locus amplified a band in Cangzhou region, but it was not stable; the band electrophoresis sites of D2mit12, D3mit22, D12wox10 and D14mit2 loci amplified in different regions. The results of D7mit26, D12wox11 and D3mit15 loci amplification were stable but polymorphic in the three regions. The results of D4mit20 loci amplification were stable but not consistent with the expected results. The genetic distance between the ten loci of norvegicus with hantavirus was not large and the genetic relationship was close.
Conclusion:
Rattus norvegicus and Mus musculus are the main rodent species in residential areas of Hebei Province. Rattus norvegicus, hamster and Mus musculus are the main rodent species in the field. Rattus norvegicus is the dominant rodent species in Hebei Province and the main source of infection of hemorrhagic fever with renal syndrome.
After comparing the Hantavirus-carrying and non-Hantavirus-carrying Rattus norvegicus at 10 microsatellite loci, no significant difference was found between them. It is not clear that Hantavirus-carrying Rattus norvegicus is related to its genetic factors.
3. By calculating the similarity coefficients, it is concluded that the relationship between Rattus norvegicus in Cangzhou and Tangshan is close, while that between Handan and Baoding, Handan and Cangzhou, Shijiazhuang and Tangshan is far, and the genetic distance is large.
Among the microsatellite loci studied, D16mit4, D18mit1 and D4mit20 were highly unstable; D2mit12, D7mit26 and D12wox10 loci were monomorphic and had little genetic difference; D3mit15, D3mit22, D12wox11 and D14mit2 loci were polymorphic and had great genetic difference.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R181.3
本文編號：2179948
[Abstract]:Objective: To understand the species composition, virulence rate and the relationship between Hantavirus carried by host animals and the genetic characteristics of the population in the epidemic focus of hemorrhagic fever with renal syndrome (HFRS) in Hebei Province, and to guide the prevention and control of the disease.
Method:
1 the investigation of the focus of the epidemic.
The lungs of mice were cut by frozen section machine, fixed by cold acetone, and screened by indirect immunofluorescence.
3 extract genomic DNA from the lungs of positive and negative mice.
According to the literature and the known microsatellite sites in GENEBANK, specific primers were searched and amplified by polymerase chain reaction (PCR), and the results of electrophoresis and observation were carried out.
5. The results of electrophoresis showed that the difference between the negative and positive samples in the same region and different regions was compared, and the genetic relationship among the populations in different regions was calculated according to the similarity coefficient.
Result:
The epidemic data in 2008 showed that Rattus norvegicus accounted for 70.39% of the total captured species, and it was the dominant species in Hebei Province. %, 20.75% and 13.21%. of rodent density in Hebei province were 2.16%, higher than that of rodent density (0.53%).
2. 331 frozen sections of rat lungs were fixed with cold acetone and detected by indirect immunofluorescence method. The positive rate was 0.30%. It was suggested that the rodents in the epidemic focus of hemorrhagic fever with renal syndrome in our province had a low virulence rate, and the rodents were Rattus norvegicus in the residential area, but not found in the field and other rodents.
The results of microsatellite amplification showed that D4mit20, D16mit4 and D18mit1 were unstable, but the amplified bands of D16mit4 were stable in Tangshan area. D18mit1 could only be amplified in positive samples of Cangzhou area, D2mit12, D3mit15, D3. There was no significant difference in the amplification results of positive and negative specimens at mit22, D7mit26, D12wox10, D12wox11 and D14mit2 loci.
The results of microsatellite amplification showed that D4mit20, D16mit4 and D18mit1 loci were very unstable, but occasionally appeared and could not be used as marker loci for genetic characteristics; D2mit12, D7mit26 and D12wox10 loci were stable and had little variation in each region; D3mit15, D3mit2 were stable and the mutation was small. 2. The genetic variations of D12wox11 and D14mit2 loci in different regions were large, showing polymorphism, and there were genetic differences among individuals.
5 Comparing the results of microsatellite amplification among Rattus norvegicus without Hantaan virus in different areas, we found that there were regional differences in D3mit15, D12wox10, D12wox11 and D14mit2 loci in Rattus norvegicus, indicating that there were genetic differences among Rattus norvegicus in different areas, and the other microsatellite loci did not show differences. The genetic distance between the populations of Rattus norvegicus in Danzhou and Baoding, Shijiazhuang and Tangshan, Cangzhou and Handan was relatively large, and the similarity coefficient between Cangzhou and Tangshan was 0.8.
6. Comparing the results of microsatellite amplification of Hantavirus in Rattus norvegicus from different regions, we found that the D16mit4 locus amplified a band in Tangshan region, the D18mit1 locus amplified a band in Cangzhou region, but it was not stable; the band electrophoresis sites of D2mit12, D3mit22, D12wox10 and D14mit2 loci amplified in different regions. The results of D7mit26, D12wox11 and D3mit15 loci amplification were stable but polymorphic in the three regions. The results of D4mit20 loci amplification were stable but not consistent with the expected results. The genetic distance between the ten loci of norvegicus with hantavirus was not large and the genetic relationship was close.
Conclusion:
Rattus norvegicus and Mus musculus are the main rodent species in residential areas of Hebei Province. Rattus norvegicus, hamster and Mus musculus are the main rodent species in the field. Rattus norvegicus is the dominant rodent species in Hebei Province and the main source of infection of hemorrhagic fever with renal syndrome.
After comparing the Hantavirus-carrying and non-Hantavirus-carrying Rattus norvegicus at 10 microsatellite loci, no significant difference was found between them. It is not clear that Hantavirus-carrying Rattus norvegicus is related to its genetic factors.
3. By calculating the similarity coefficients, it is concluded that the relationship between Rattus norvegicus in Cangzhou and Tangshan is close, while that between Handan and Baoding, Handan and Cangzhou, Shijiazhuang and Tangshan is far, and the genetic distance is large.
Among the microsatellite loci studied, D16mit4, D18mit1 and D4mit20 were highly unstable; D2mit12, D7mit26 and D12wox10 loci were monomorphic and had little genetic difference; D3mit15, D3mit22, D12wox11 and D14mit2 loci were polymorphic and had great genetic difference.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R181.3
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