云南大理片形吸蟲掃描電鏡觀察和分子生物學(xué)鑒定
發(fā)布時(shí)間：2018-10-12 11:44
【摘要】：[目的]觀察云南大理片形吸蟲頭錐部分(口吸盤、腹吸盤、生殖孔、體棘)的超微結(jié)構(gòu),用分子生物學(xué)方法對(duì)該地區(qū)片形吸蟲的蟲種進(jìn)行鑒定,為云南大理片形吸蟲的生物學(xué)特性及遺傳變異提供基礎(chǔ)信息。[方法]1.云南大理片形吸蟲部分結(jié)構(gòu)的掃描電鏡觀察:從大理泰興農(nóng)貿(mào)市場(chǎng)自然感染片形吸蟲的4頭黃牛肝膽管中獲得了片形吸蟲各10條,用生理鹽水沖洗去血跡后各自分裝到裝滿生理鹽水的玻璃瓶中,依次標(biāo)記為1、2、3、4號(hào)。從4號(hào)瓶中選取兩條較完整的蟲體編號(hào)為A、B號(hào),從3號(hào)瓶選取了兩條較完整的蟲體編號(hào)為C、D號(hào)。剪取四條蟲體頭錐部分經(jīng)冷藏過夜處理后按照制作掃描電鏡標(biāo)本的步驟:取材、固定、脫水、包埋、切片、染色后放在電子顯微鏡下進(jìn)行觀察。2.提取片形吸蟲成蟲基因組DNA:1號(hào)瓶中獲得較完整蟲體6條,編號(hào)為1-6號(hào);2號(hào)瓶中獲得蟲體2條,編號(hào)7-8號(hào),取這些蟲體前端1/3部分,生理鹽水沖洗,再用超純水沖洗4到5遍,裝于已消毒的1.5ml離心管。用手持式研磨儀搗碎蟲體,將所得組織碎片參照TaKaRa MiniBEST Universal Genomic DNA Extraction KitVer.5.0試劑盒說明書上的方法提取片形吸蟲成蟲DNA,用核酸定量?jī)x檢測(cè)8個(gè)樣品的DNA濃度。3.PCR擴(kuò)增和測(cè)序:擴(kuò)增片形吸蟲的核糖體ITS-2基因片段、線粒體cox1部分基因片段及線粒體nad5部分基因片段,將所得擴(kuò)增產(chǎn)物送至大連寶生物公司測(cè)序。4.序列分析:用DNAStar7.0軟件將所得序列與之前報(bào)道的標(biāo)準(zhǔn)序列進(jìn)行相似性比較。[結(jié)果]1.除C號(hào)蟲體未見明顯的生殖孔以外,其他三條片形吸蟲都具有片形吸蟲的典型結(jié)構(gòu):口吸盤、腹吸盤、生殖孔以及遍布蟲體的體棘�？谖P直徑在310μm-540μm之間,腹吸盤直徑在450μm-600μm之間。對(duì)這些蟲體的口、腹吸盤放大后顯示具有兩種形式的乳突:圓錐形乳突和纖毛樣乳突。將蟲體部分體棘高倍放大后發(fā)現(xiàn)其呈現(xiàn)橫向生長(zhǎng)于蟲體表面的具有基底較寬,端部較窄的圓弧狀結(jié)構(gòu),端部生長(zhǎng)有鋸齒樣結(jié)構(gòu)。2.8條片形吸蟲進(jìn)行分子生物學(xué)鑒定后顯示:其中2條經(jīng)PCR擴(kuò)增及瓊脂糖凝膠電泳后顯示大片形吸蟲的目的條帶,因此這兩條鑒定為大片形吸蟲;另外6條經(jīng)PCR擴(kuò)增及瓊脂糖凝膠電泳后顯示肝片形吸蟲的目的條帶,這6條鑒定為肝片形吸蟲。3.PCR擴(kuò)增云南大理片形吸蟲的線粒體cox1基因部分序列測(cè)序顯示其種內(nèi)分歧度為0.9%-2.3%,種間分歧度3.0%-10.0%;線粒體nad5基因部分序列其種內(nèi)分歧度為0.9%-1.2%,種間分歧度為 5.0%-13.0%。[結(jié)論]1.云南大理片形吸蟲部分結(jié)構(gòu)的掃描電鏡結(jié)果顯示四條片形吸蟲有三條是肝片形吸蟲,一條是大片形吸蟲。除一條片形吸蟲未觀察到明顯的生殖孔以外,其余三條片形吸蟲都具有口腹吸盤、生殖孔,且蟲體表面均生長(zhǎng)一些致密的體棘,而且口吸盤處體棘比腹吸盤處致密。2.在以核糖ITS-2基因?yàn)榉诸愡z傳標(biāo)記的研究基礎(chǔ)之上,云南大理存在兩種片形吸蟲:肝片形吸蟲與大片形吸蟲。3.通過對(duì)這8條片形吸蟲樣品的核糖體ITS-2基因和線粒體cox1、nad5基因部分序列進(jìn)行測(cè)序顯示該地區(qū)片形吸蟲之間呈現(xiàn)種間差異大于種內(nèi)差異的特點(diǎn)。
[Abstract]:[Objective] To observe the ultrastructure of the cone part (mouth suction cup, abdomen suction cup, hole and body spine) of Fasciola paragonimus in Yunnan Province, and to identify the insect species of the Fasciola paragonimus by molecular biology method. To provide basic information for the biological characteristics and genetic variation of paragonimus yunnanensis.[Method] 1. Scanning electron microscope (SEM) of the partial structure of paragonimiasis in Yunnan Province: 10 pieces of Fasciola paragonimus were obtained from 4 Yellow Cattle Hepatobiliary Tubes of the Natural Infection Tablets of Taixing Agricultural Trade Market. After washing the blood with physiological saline, they were separately packed into glass bottles filled with physiological saline. Mark 1, 2, 3, 4 in sequence. From bottle No. 4, select two more complete bottles numbered A and B, and select two more complete numbers from No. 3 bottle to be C, D. The specimens were collected, fixed, dehydrated, embedded, sectioned, stained and observed under an electron microscope after cold storage overnight. The genomic DNA of adult adult worm was extracted: 1 bottle, 6 with no number 1-6, 2 in bottle 2, 7-8 No. 7-8, washed with physiological saline, flushed with ultrapure water for 4 to 5 times, and contained in sterilized 1. 5ml centrifuge tube. The DNA of the paragonimus paragonimus is extracted by a hand-held grinder, and the DNA concentration of 8 samples is detected by a nucleic acid quantitative instrument by referring to the DNA of the TaKaRa MiniBEST Universal Genomeic DNA Extraction KitVer. 5. 0 kit specification. The PCR amplification and sequencing are carried out to amplify the ribosomal ITS-2 gene fragment of the slice-shaped paragonimus, mitochondrial cox1 partial gene fragment and mitochondrial nad5 partial gene fragment, and the obtained amplified product is sent to Dalian Bao biological company for sequencing. Sequence analysis: The obtained sequence was compared with the previously reported standard sequence using DNAStar7.0 software.[Results] 1. The other three tablets have the typical structure of the paragonimus except that the C No. 48 is not seen in the obvious maxillary foramen: the mouth sucker, the belly sucker, the foramen magnum, and the body spine spread all over the body. The diameter of the mouth sucker is between 310. m u.m and 540. m u.m, and the diameter of the abdominal suction disc is between 450. m u.m and 600. m Two forms of mastoid, conical mastoid and ciliated mastoid, were displayed after enlargement of the abdominal suction cups. it is found that it has a wider base and a narrower end part, and the end part grows with sawtooth-like structure. Two of them were amplified by PCR and agarose gel electrophoresis to show the target bands of large-scale paragonimus, so the two were identified as large-scale paragonimus, and 6 were amplified by PCR and agarose gel electrophoresis to show the objective bands of the Fasciola paragonimus. The mitochondrial cox1 gene partial sequence of paragonimus paragonimus was amplified by PCR, and the difference between species was 0. 9% -2. 3%, the difference between species was 3.0%-10.0%, and the divergence of mitochondrial nad5 gene partial sequence was 0. 9%-1. 2%, and the difference between species was 5.0%-13.0%.[Conclusion] 1. The scanning electron microscope (SEM) of the partial structure of paragonimiasis in Yunnan Province showed that there were three pieces of paragonimiasis, one of which was a large piece of paragonimus. except that one piece of paragonimus was not observed, the other three pieces of paragonimus had an abdominal suction cup and a buccal cavity, and a compact body was grown on the surface of the mouth, and the body spine at the mouth sucker was denser than that at the abdominal suction cup. On the basis of the research on the classification of genetic markers with ribose ITS-2 gene, there are two kinds of paragonimus in Yunnan Province: Fasciola paragonimus and large-scale paragonimus. By sequencing the ribosomal ITS-2 gene and mitochondrial cox1, nad5 gene partial sequences of the 8 pieces of paragonimiasis, the differences between species and species in the region were found to be more than that of intraspecific differences.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R383.2
本文編號(hào)：2265982
[Abstract]:[Objective] To observe the ultrastructure of the cone part (mouth suction cup, abdomen suction cup, hole and body spine) of Fasciola paragonimus in Yunnan Province, and to identify the insect species of the Fasciola paragonimus by molecular biology method. To provide basic information for the biological characteristics and genetic variation of paragonimus yunnanensis.[Method] 1. Scanning electron microscope (SEM) of the partial structure of paragonimiasis in Yunnan Province: 10 pieces of Fasciola paragonimus were obtained from 4 Yellow Cattle Hepatobiliary Tubes of the Natural Infection Tablets of Taixing Agricultural Trade Market. After washing the blood with physiological saline, they were separately packed into glass bottles filled with physiological saline. Mark 1, 2, 3, 4 in sequence. From bottle No. 4, select two more complete bottles numbered A and B, and select two more complete numbers from No. 3 bottle to be C, D. The specimens were collected, fixed, dehydrated, embedded, sectioned, stained and observed under an electron microscope after cold storage overnight. The genomic DNA of adult adult worm was extracted: 1 bottle, 6 with no number 1-6, 2 in bottle 2, 7-8 No. 7-8, washed with physiological saline, flushed with ultrapure water for 4 to 5 times, and contained in sterilized 1. 5ml centrifuge tube. The DNA of the paragonimus paragonimus is extracted by a hand-held grinder, and the DNA concentration of 8 samples is detected by a nucleic acid quantitative instrument by referring to the DNA of the TaKaRa MiniBEST Universal Genomeic DNA Extraction KitVer. 5. 0 kit specification. The PCR amplification and sequencing are carried out to amplify the ribosomal ITS-2 gene fragment of the slice-shaped paragonimus, mitochondrial cox1 partial gene fragment and mitochondrial nad5 partial gene fragment, and the obtained amplified product is sent to Dalian Bao biological company for sequencing. Sequence analysis: The obtained sequence was compared with the previously reported standard sequence using DNAStar7.0 software.[Results] 1. The other three tablets have the typical structure of the paragonimus except that the C No. 48 is not seen in the obvious maxillary foramen: the mouth sucker, the belly sucker, the foramen magnum, and the body spine spread all over the body. The diameter of the mouth sucker is between 310. m u.m and 540. m u.m, and the diameter of the abdominal suction disc is between 450. m u.m and 600. m Two forms of mastoid, conical mastoid and ciliated mastoid, were displayed after enlargement of the abdominal suction cups. it is found that it has a wider base and a narrower end part, and the end part grows with sawtooth-like structure. Two of them were amplified by PCR and agarose gel electrophoresis to show the target bands of large-scale paragonimus, so the two were identified as large-scale paragonimus, and 6 were amplified by PCR and agarose gel electrophoresis to show the objective bands of the Fasciola paragonimus. The mitochondrial cox1 gene partial sequence of paragonimus paragonimus was amplified by PCR, and the difference between species was 0. 9% -2. 3%, the difference between species was 3.0%-10.0%, and the divergence of mitochondrial nad5 gene partial sequence was 0. 9%-1. 2%, and the difference between species was 5.0%-13.0%.[Conclusion] 1. The scanning electron microscope (SEM) of the partial structure of paragonimiasis in Yunnan Province showed that there were three pieces of paragonimiasis, one of which was a large piece of paragonimus. except that one piece of paragonimus was not observed, the other three pieces of paragonimus had an abdominal suction cup and a buccal cavity, and a compact body was grown on the surface of the mouth, and the body spine at the mouth sucker was denser than that at the abdominal suction cup. On the basis of the research on the classification of genetic markers with ribose ITS-2 gene, there are two kinds of paragonimus in Yunnan Province: Fasciola paragonimus and large-scale paragonimus. By sequencing the ribosomal ITS-2 gene and mitochondrial cox1, nad5 gene partial sequences of the 8 pieces of paragonimiasis, the differences between species and species in the region were found to be more than that of intraspecific differences.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R383.2
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