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目的为进一步了解我国斑点热群立克次体存在的多样性,发现可能存在的斑点热群立克次体新成员、潜在媒介和动物宿主。方法建立了扩增斑点热群立克次体190kDa rOmpA基因片段的PCR检测、鉴定方法,并用此方法检测了采自福建省和内蒙古自治区的蜱、动物和人血液标本。对扩增于越原血蜱、森林革蜱和FNH97未鉴定菌株的PCR产物采用PHYLIP软件包进行了序列分析。同时,为了寻找特异的斑点热群立克次体分类检测方法,建立了针对四种斑点热群立克次体的半巢式PCR方法,并对斑点热立克次体阳性标本进行了分类检测。结果采用 190kDa rOmpA.701/70p引物可以从7株斑点热群立克次体中的6株扩增出外膜蛋白A基因片段(小蛛立克次体除外)。并从采自福建省和内蒙古自治区的多种蜱及野生动物、人血液标本中扩增出了斑点热立克次体DNA片段,其中越原血蜱、卵形硬蜱、中华硬蜱、豪猪血蜱、森林革蜱、野鼠血块和人群血块的阳性率分别为15.69％、56.94％、8.70％、7.70％、43.56％、82.51％和0.98％。对来自越原血蜱和森林革蜱以及FNH97菌株的斑点热立克次体190kDa外膜蛋白A630bp左右核苷酸片段序列分析和推测的氨基酸序列分析结果表明:福建越原血蜱立克次体(福建立克次体)核苷酸序列与日本立克次体的该序列同源性最高(94％)。推测氨基酸序列同源性也与该立克次体最高(89％);内蒙古森林革蜱立克次体(森林革蜱立克次体)核苷酸序列与扇头蜱立克次的该序列同源性最高(97％),推测氨基酸序列的同源性也最高(95％)。遗传发育分析,这两种立克次体分别与日本立克次体和扇头蜱立克次体均为同一个分支。序列中限制性核酸内切酶的位点也显示了对应的相似性。但是它们的核苷酸和推测的氨基酸序列与康氏立克次体和西伯利亚立克次体以及内蒙古立克次体(HA-91)差别较大。提示,这两种蜱携带的立克次体可能是我国尚未发现的斑点热群立克欢体新成员。用初步分类引物对蜱标本、血液标本检测结果显示:以“福建立克次体”序列设计的引物检测越原血蜱阳性率为8.49％,卵形硬蜱阳性率为20.83％,中华硬蜱阳性率为4.35％,豪猪血蜱为阴性;以西伯利亚立克次体序列设计的引物检测卵形硬蜱阳性率为 24.39％,越原血蜱阳性率为 5.56％,中华硬蜱和豪猪血蜱均为阴性。以内蒙革蜱立克次体序列设计的引物扩增森林革蜱阳性率为43.56％。结论通过本次研究,在以下方面获得了新的认识:越原血蜱、卵形硬蜱、豪猪血蜱和中华硬蜱是福建南方蜱传斑点热立克次体的媒介或潜在媒介,其中卵形硬蜱、豪猪血蜱和中华硬蜱为我国首次证实的携带斑点热立克次体;福建的越原血蜱和内蒙古的森林革蜱分别携带一种未知斑点热群立克次体,并分别与日本立克次体和肩头蜱立克次体近缘;取自斑点热立克次体rOmpA基因的引物用于PCR,作为一种快速简便手段可直接用于斑点热立克次体初步分类和分子流行病学调查;我国可能存在斑点热群立克次体的多个成员及其自然疫源地。
Aim To further understand the diversity of Rickettsia genus in our country, we found possible new members, potential vectors and animal hosts of Rickettsia genus. Methods A PCR detection and identification method for the 190 kDa rOmpA gene fragment of Rickettsia plague was established and used to detect tick, animal and human blood samples collected from Fujian and Inner Mongolia autonomous regions. PCR products amplified from P. vivax, F. graminis and unidentified strains of FNH97 were sequenced using the PHYLIP software package. At the same time, in order to find out the specific speckle detection method of Rickettsia, a semi-nested PCR method was established for four speckles of Rickettsia rickettsi and the positive samples of Rickettsia glabrata were classified and tested . Results A 190 kDa rOmpA.701 / 70p primer was used to amplify the outer membrane protein A gene (except for the small spider Rickettsia) from 6 of 7 spotted hot group rickettsia. In addition, spotted hot rickettsia DNA fragments were amplified from a variety of tick and wild animal blood samples collected from Fujian Province and Inner Mongolia Autonomous Region. Among them, Acipenser sinensis, Ixodes scapularis, Ixodes sinensis, porcupine The positive rates of blood-borne tick, forest tick, wild rat and blood clot were 15.69%, 56.94%, 8.70%, 7.70%, 43.56%, 82.51% and 0.98%, respectively. Sequence analysis and deduced amino acid sequence analysis of A630bp nucleotide fragment of 190kDa outer membrane protein from the spotted fever tick and forest flies tick and FNH97 strain showed that: (Fujian Rickettsia) nucleotide sequence and the Japanese Rickettsia the highest homology (94%). The amino acid sequence homology was also found to be the highest (89%) for this rickettsia. The nucleotide sequence of Rickettsia korshoffii The homology was the highest (97%), suggesting the highest amino acid sequence homology (95%). Genetic analysis of development, the two rickettsia respectively with the Japanese Rickettsiae and the first tick rickettsia are the same branch. Sites of restriction endonucleases in the sequence also showed corresponding similarities. However, their nucleotide and putative amino acid sequences differ greatly from those of R. rhamnosus and S. rickettsii and Inner Mongolia rickettsia (HA-91). It is suggested that the Rickettsia species carried by these two ticks may be the new members of Rickettsia, a spotted fever group that has not been found in our country. The test results of ticks and blood samples by the preliminary classification primers showed that the positive rate of the whole blood of ticks was 8.49%, the percentage of the eggs was 20.83% The positive rate was 4.35%. The positive rate was 4.36%, and the positive rate was 5.56%. The positive rate of Cyanidium Ixodes was 24.39% All negative. The positive rate of forest ticks was 43.56% based on the primers designed by the sequence of Rickettsia in Inner Mongolia. CONCLUSIONS This research has led to new understandings in the following areas: Acanthamoebae, Ixodes tricuspidata, Porcupine Ichthyophthis and Ixodes sinensis are vectors or potential vectors for tick-spotted fever ticks in southern Fujian, where Ixodes ovatus, porcupine hedgehog and Chinese Ixodes were the first confirmed spotted fever rickettsia in our country. Fujian wild ticks and forest ticks in Inner Mongolia carried an unknown spotted fever group Rickettsia, And were closely related to Rickettsia and Rickettsia tricuspidae, respectively; primers from the rOmpA gene of Rickettsia grisea were used for PCR as a rapid and simple means for direct use in the detection of Rickettsia glabra Preliminary classification and molecular epidemiological investigation; there may be multiple members of the speckle fever group rickettsia and their natural foci.