中华诊断学电子杂志
中華診斷學電子雜誌
중화진단학전자잡지
2014年
4期
270-274
,共5页
李瑾%徐超%魏庆宽%肖婷%孔祥礼%王用斌%张本光%魏艳彬%赵长磊%黄炳成
李瑾%徐超%魏慶寬%肖婷%孔祥禮%王用斌%張本光%魏豔彬%趙長磊%黃炳成
리근%서초%위경관%초정%공상례%왕용빈%장본광%위염빈%조장뢰%황병성
疟原虫,三日%核糖体亚单位%RNA,核糖体%聚合酶链式反应,套式%诊断
瘧原蟲,三日%覈糖體亞單位%RNA,覈糖體%聚閤酶鏈式反應,套式%診斷
학원충,삼일%핵당체아단위%RNA,핵당체%취합매련식반응,투식%진단
Plasmodium malariae%Ribosome subunits%RNA,ribosomal%Polymerase chain reaction,nested%Diagnosis
目的：应用套式聚合酶链式反应（PCR）扩增小亚单位核糖体核糖核酸（SSUrRNA）基因诊断三日疟原虫感染，以减少三日疟的漏诊和误诊。方法分别提取可疑三日疟患者抗凝血 DNA和对照间日疟、恶性疟患者抗凝血 DNA，以此为模板，用疟原虫属特异性引物进行第一轮扩增；然后以第一轮扩增产物为模板，用4种疟疾的种特异性引物进行第二轮扩增，比较扩增出的18 SSU rRNA基因片段的大小，并对目的片段进行测序鉴定。结果经属特异性引物 PCR 扩增后，3个样本均出现大小约为1200bp 的条带。经种特异性引物 PCR 扩增后，间日疟及恶性疟确诊样本均扩增出相应的120bp 和205bp 特异性条带；可疑患者样本仅在用三日疟原虫种特异性作引物时扩增出144bp 特异条带，与理论值相符，与 Genbank 标准序列对比显示，扩增片段大小及测序结果均完全正确。证实患者感染三日疟原虫。结论利用小亚单位核糖体核糖核酸基因片段三日疟种特异引物进行扩增可以用于诊断三日疟原虫感染。
目的：應用套式聚閤酶鏈式反應（PCR）擴增小亞單位覈糖體覈糖覈痠（SSUrRNA）基因診斷三日瘧原蟲感染，以減少三日瘧的漏診和誤診。方法分彆提取可疑三日瘧患者抗凝血 DNA和對照間日瘧、噁性瘧患者抗凝血 DNA，以此為模闆，用瘧原蟲屬特異性引物進行第一輪擴增；然後以第一輪擴增產物為模闆，用4種瘧疾的種特異性引物進行第二輪擴增，比較擴增齣的18 SSU rRNA基因片段的大小，併對目的片段進行測序鑒定。結果經屬特異性引物 PCR 擴增後，3箇樣本均齣現大小約為1200bp 的條帶。經種特異性引物 PCR 擴增後，間日瘧及噁性瘧確診樣本均擴增齣相應的120bp 和205bp 特異性條帶；可疑患者樣本僅在用三日瘧原蟲種特異性作引物時擴增齣144bp 特異條帶，與理論值相符，與 Genbank 標準序列對比顯示，擴增片段大小及測序結果均完全正確。證實患者感染三日瘧原蟲。結論利用小亞單位覈糖體覈糖覈痠基因片段三日瘧種特異引物進行擴增可以用于診斷三日瘧原蟲感染。
목적：응용투식취합매련식반응（PCR）확증소아단위핵당체핵당핵산（SSUrRNA）기인진단삼일학원충감염，이감소삼일학적루진화오진。방법분별제취가의삼일학환자항응혈 DNA화대조간일학、악성학환자항응혈 DNA，이차위모판，용학원충속특이성인물진행제일륜확증；연후이제일륜확증산물위모판，용4충학질적충특이성인물진행제이륜확증，비교확증출적18 SSU rRNA기인편단적대소，병대목적편단진행측서감정。결과경속특이성인물 PCR 확증후，3개양본균출현대소약위1200bp 적조대。경충특이성인물 PCR 확증후，간일학급악성학학진양본균확증출상응적120bp 화205bp 특이성조대；가의환자양본부재용삼일학원충충특이성작인물시확증출144bp 특이조대，여이론치상부，여 Genbank 표준서렬대비현시，확증편단대소급측서결과균완전정학。증실환자감염삼일학원충。결론이용소아단위핵당체핵당핵산기인편단삼일학충특이인물진행확증가이용우진단삼일학원충감염。
Objective To diagnose the imported plasmodium malariae cases with nested-PCR amplification of 1 8SSUrRNA gene.Methods Plasmodium DNA was extracted from anticoagulant blood as template with the plasmodium gene-specific primers in the first round of amplification,then the products of the first round were amplified as the second round template.The results of different subgroups of plasmodium were acquired.The target fragment of PCR was used for species identification and sequencing,comparing with the control sample of P.vivax and P.falciarum.Results By using the plasmodium gene-specific primers amplification,the fragments were amplified from the three samples with 1 200 bp.By using the interspecific primers amplification,specific fragments were amplified from the blood samples of P.vivax and P.falciarum with 1 20 bp and 205 bp respectively,and 1 44 bp from the suspicious patient with P.malariae specific primers.The suspicious patients did not have the specific fragment of P.ovale.The product of the suspicious patients was sequenced and compared with standard sequence registered in Genbank by blast.The comparison results showed that the fragment size and product sequence were in accordance with the reference.The results showed that nested PCR was more objective than the traditional microscopic examination.Conclusions The results indicate that nested PCR can be used to detect plasmodium with high specificity and identify plasmodium at the species level when microscopy is equivocal.The specific primers to amplify 1 8 SSUrRNA gene fragment can be used to diagnose plasmodium malariae infection.
