环介导等温扩增检测恶性疟原虫与其他疟原虫的效果评价

目的采用环介导等温扩增(LAMP)方法检测疟原虫DNA,并评价其检测效果。方法采集2013-2018年从疟疾流行区回国人员中确诊为疟疾患者的滤纸血样。根据疟原虫的18S r RNA序列,使用PrimerExplorer V5软件设计恶性疟原虫与非恶性疟原虫LAMP引物,建立疟原虫LAMP检测方法,进行敏感性、特异性、灵敏度和扩增效率评价,并与巢式PCR比较。检测钙黄绿素指示剂对LAMP反应的影响。结果LAMP和巢式PCR各检测96份疟疾患者血样(47份恶性疟血样和49份其他疟疾血样),均检出93份疟原虫阳性。LAMP检测结果显示,恶性疟血样均被检出,非恶性疟假阴性率为2.1%, 10份利什曼原虫玻片血、 8份日本血吸虫感染兔血和38份健康人血均为阴性,与巢式PCR检测结果具有极高的一致性(Kappa值=0.956)。LAMP检测恶性疟原虫、间日疟原虫、卵形疟原虫、三日疟原虫的灵敏度分别为25、 3、 4、 2个疟原虫/μl血。扩增效率检测结果显示,4种疟原虫LAMP反应出现浊度时间(Tt值)均小于60 min,速率曲线峰值(Df)达0.14以上,其中恶性疟原虫扩增效率较高,Tt值为20.4 min, Df达0.16。可视化LAMP检测结果显示,钙黄绿素指示剂对不同种疟原虫LAMP反应延时约8～22 min, Df下降约21%～35%。结论 LAMP技术检测疟原虫有高度敏感性、特异性和灵敏度,且可视,具有在现场及基层医疗机构应用前景。     

多重PCR快速检测恶性疟和间日疟的研究

目的 建立一种简便快速、能同时检测恶性疟和间日疟的核酸检测方法。方法 针对两种疟原虫18S rRNA基因设计2对(3条引物),优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的多重PCR。并进行最低检测限确定和临床标本检测,以镜检法为金标准分析灵敏度和特异度等指标。结果 该方法可扩增出431 bp(恶性疟原虫)和341 bp(间日疟原虫)基因片段,最低检测限为10²copies/反应,检测临床标本的结果与镜检法无差别(P＞0.05),敏感度为93.55%,特异度为70.83%,阳性预测值为89.23%,阴性预测值为80.95%。结论 所建立的多重PCR方法可快速检测疟疾感染并鉴别分型,灵敏度高,值得推广。     

巢式PCR法在疟疾检测及虫种鉴别中的应用

根据疟原虫小亚单位核糖体核糖核酸(SSU rRNA)基因序列设计疟原虫通用型和种特异性的引物,对60份血样进行巢式PCR检测及虫种鉴定,并与血样的吉氏染色镜检结果进行比较。巢式PCR检出40份疟原虫阳性血样,其中22份为恶性疟原虫(Plasmodium falciparum)阳性、13份为间日疟原虫(P.vivax)阳性、3份为恶性疟原虫和间日疟原虫混合感染、1份为卵形疟原虫阳性(P.ovale)、1份未能分型。与镜检结果一致的血样为46份,占76.7%(46/60),其中恶性疟原虫阳性18份、间日疟原虫阳性11份和阴性17份。将两种检测结果不一致的血样进行扩增片段序列测定和实时荧光PCR分析,检测结果均与巢式PCR结果一致。卵形疟原虫阳性血样扩增片段的序列分析结果显示,该序列与卵形疟原虫SSU rRNA基因序列(GenBank登录号DQ845247)的对应部分同源性为100%,证实该病例为输入性卵形疟原虫感染病例。     

PCR检测疟疾混合感染的现场应用研究

目的　评价PCR检测技术在间日疟与恶性疟混合感染区诊断疟疾的现场应用价值。　方法　采集海南省疟疾混合感染流行区 3 0 4份滤纸干血滴样本 ,根据红内期疟原虫SSUrRNA基因序列 ,设计合成 3条引物 ,采用PCR技术在同一反应体系中扩增出间日疟原虫和恶性疟原虫不同的DNA片段 ,检测现场所采样本中的间日疟原虫或恶性疟原虫DNA ;同时与镜检法进行比较。　结果　在 3 0 4份样本中 ,PCR法阳性 15份 ,其中间日疟 7份 ,恶性疟 8份 ;镜检法阳性11份 ,其中间日疟 6份 ,恶性疟 5份。镜检阳性的样本PCR均为阳性 ;镜检阴性而PCR阳性的 4份样本 ,其扩增产物经限制性酶切鉴定 ,证实为间日疟原虫或恶性疟原虫DNA。　结论　此PCR检测体系灵敏、特异 ,对诊断或鉴别诊断间日疟原虫和恶性疟原虫混合感染具有实用价值。     

PCR检测疟疾混合感染的现场应用研究

目的 评价PCR检测技术在间日疟与恶性疟混合感染区诊断疟疾的现场应用价值。方法 采集海南省疟疾混合感染流行区304份滤纸干血滴样本，根据红内期疟原虫SSUrRNA基因序列，设计合成3条引物．采用PCR技术在同一反应体系中扩增出间日疟原虫和恶性疟原虫不同的DNA片段，检测现场所采样本中的间日疟原虫或恶性疟原虫DNA；同时与镜检法进行比较。结果 在304份样本中，PCR法阳性15份，其中间日疟7份．恶性疟8份；镜检法阳性11份，其中间日疟6份，恶性疟5份。镜检阳性的样本PCR均为阳性；镜检阴性而PCR阳性的4份样本，其扩增产物经限制性酶切鉴定，证实为间日疟原虫或恶性疟原虫DNA。结论 此PCR检测体系灵敏、特异．对诊断或鉴别诊断间日疟原虫和恶性疟原虫混合感染具有实用价值。     

聚合酶链反应技术与镜检法诊断间日疟的对比研究

目的　评价聚合酶链反应技术在间日疟诊断中的应用价值。　方法　根据红内期疟原虫 SSUr RNA编码基因序列 ,设计合成 1对引物 ,采用聚合酶链反应技术 ,扩增检测“四热”病人血样中间日疟原虫 DNA;同时作厚血膜镜检疟原虫。　结果　从间日疟患者血样中扩增出 341bp的 DNA片段 ,与预期的扩增片段大小相符 ,而正常人血样及空白对照无特异性条带 ;对于 2 34份“四热”病人血样 ,PCR法检出 16份阳性 ,阳性率为 6 .8% ;厚血膜镜检 13份阳性 ,阳性率为 5 .6 % ;两种方法相比差异无显著性 (P>0 .0 5 ) ;对于 3份 PCR阳性而镜检法阴性的血样 ,重新作 PCR,结果仍然为阳性。以镜检法为标准 ,PCR法的灵敏度为 10 0 % ,特异度为 98.6 %。　结论　PCR技术灵敏特异 ,可替代镜检法用于间日疟感染的临床及现场检测。     

巢式PCR技术在输入性疟疾监测中的应用

目的评价巢式PCR技术在高疟区回国人员疟疾监测中的应用价值。方法收集2007-2009年自非洲、东南亚等疟疾流行区回国人员中疟疾现症患者及其他无症状高危人群的滤纸血,采用巢式PCR技术检测疟原虫ssRNA基因,并与血片镜检结果进行比对。结果巢式PCR检测53例血检确诊的疟疾现症患者均为阳性,两法的阳性符合率为100%,其中PCR检出恶性疟、间日疟混合感染6例,高于镜检检出的3例。检测疟疾流行区返回的高危人群157人,镜检阳性3例,阳性率1.91%;巢式PCR阳性5例,阳性率为3.18%;其中,镜检阳性、巢式PCR阴性的1例,镜检阴性、巢式PCR阳性的3例,两种方法的阳性符合率为66.7%,阴性符合率为98.1%,两法检测结果一致的血样占检测血样的97.5%。结论巢式PCR技术对输入性疟疾的监测具有实用价值。     

捕获和连接探针PCR与巢氏PCR检测疟原虫效果的比较

目的了解捕获与连接探针PCR(CLIP-PCR)对疟原虫的确认诊断效果。方法云南省疟疾诊断参比实验室提供的血涂片镜检阴性和阳性者滤纸血样各70份,分别进行盲法CLIP-PCR和巢氏PCR检测,以巢氏PCR为金标准评价CLIP-PCR的检测效果及检测耗时,血样梯度稀释后测定CLIP-PCR最低检出阈值。结果检测140份血样,2种PCR检出假阳性和假阴性各1例,CLIP-PCR检测疟原虫的敏感度、特异度、阳性预测值、阴性预测值和诊断准确度均为98.57%,kappa值为0.97;CLIP-PCR耗时13.91 h,较巢氏PCR耗时23.47 h缩短40.73%;梯度稀释后重复检测8次,≥3.2个虫/μl血检出率100%,0.32个虫/μl血检出率50%。结论 CLIP-PCR检测疟原虫效果与巢氏PCR相同,重复性良好,更适用于大规模人群筛查。     

"新吉尔"疟原虫快速检测试剂盒检测效果评价

目的采用疟疾厚薄血片镜检法(金标准)和"新吉尔"疟原虫抗原检测试剂盒(胶体金法)进行平行检测,评价该疟疾快速检测试剂盒的检测效果。方法收集948份血样,采用镜检作为金标准与新吉尔快速诊断试剂盒进行盲法平行检测,计算试剂盒检测结果的灵敏度、特异性和诊断准确性,分析两种检测方法检测结果的一致性。结果共检测948份血样,其中镜检疟原虫阳性416份。以镜检法结果为标准,"新吉尔"疟原虫检测试剂盒检测灵敏度为96.88%,特异性为100%,诊断准确性为98.63%,一致性系数kappa为0.9721。其中检测间日疟的灵敏度为94.76%,特异度为100%,诊断准确性为98.62%,一致性系数kappa为0.9638;检测恶性疟灵敏度达98.41%,特异度为100%,诊断准确性为99.58%,一致性系数kappa达0.9892。间日疟原虫的检出下限介于105.19-157.94个/?l之间,恶性疟原虫的检测下限介于77.76-103.68个/?l之间。结论与镜检法相比,新吉尔疟原虫检测试剂盒具有较高的灵敏度、特异度和一致性,其中对恶性疟检测的灵敏度和一致性略高于间日疟,但是在原虫密度较低情况下漏检较多。该试剂盒可用于不具备开展镜检的基层医疗机构进行疟疾的快速检测和初筛,但不建议用于健康人群或无症状人群的筛查和检测。     

镜检、抗原检测和核酸检测方法在疟疾诊断中的应用比较

目的 比较镜检、抗原检测(RDT)和核酸检测(PCR)三种方法对疟原虫的检测效果,为基层选择合适的 诊断方法提供依据。 方法 收集腾冲市 2015-2018 年发热病人的血样进行疟疾检测,以确诊结果为标准,对比分析镜检、RDT 和 PCR 三种疟疾检测方法的敏感性、特异性、阳性预测值、阴性预测值等指标。 结果 610 份血样中,阴性 295 份,阳性 315 份,其中恶性疟 67 份、间日疟 245 份、混合感染 2 份、三日疟 1 份。 与确诊结果比较,镜检、RDT 和 PCR 的灵敏度分别为 95. 87%、94. 60%和 99. 37%,特异度均为 100%;假阴性率分别为 4. 13%、5. 40%和 0. 63%,阴性预 测值分别为 95. 78%、94. 55%和 99. 33%;假阳性率均为 0,阳性预测值均为 100%;三种方法与确诊结果的总符合率分别 为 97. 87%、97. 70%和 99. 67%,Kappa 检验结果显示均与确诊结果高度一致(P 均<0. 001);对单一虫种恶性疟的检测, 镜检、RDT 和 PCR 的符合率分别为 88. 06%、100%和 97. 01%;对其他三种疟原虫检测的符合率,分别为 97. 97%、 93. 9%和 100%。 结论 三种检测方法均具有较高的敏感性和特异性,但综合考虑当前防治工作实际,抗原检测(RDT) 更适宜在基层推广和使用。     

Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: Loop-mediated isothermal amplification for malaria diagnosis

This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к = 0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к = 0.64) and poor to fair for saliva LAMP and urine LAMP (к = 0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.     

Detection of Plasmodium vivax infection in the Republic of Korea by loop-mediated isothermal amplification (LAMP)

Loop-mediated isothermal amplification (LAMP) is a novel technique that rapidly amplifies target DNA in isothermal conditions. In a previous study, the sensitivities and specificities of LAMP, microscopy, and nested PCR were compared in the context of rapid malaria detection. In the present study, LAMP detected vivax malaria parasites in 115 of 117 microscopically positive samples (sensitivity, 98.3%; 95% CI, 97.4-100%), which agreed well with the nested PCR results (sensitivity, 99.1%; 95% CI: 96.0-100%). No positive cases of malaria were detected by LAMP or nested PCR in 50 consecutive feverish patients other than malaria from malaria endemic areas. LAMP performed on DNA extracted from heat-treated blood had a sensitivity of 93.3% (28/30, 95% CI: 84.4-100%) and specificity of 100% (30/30, 95% CI: 100%). The present study shows that LAMP based assays have high sensitivity, specificity, and amplification efficiencies for Plasmodium vivax detection. The authors recommend that LAMP can be considered as a rapid nucleic acid amplification assay for the molecular diagnosis of P. vivax in both clinical laboratories and malaria clinics in areas where vivax malaria is endemic.     

Evaluation of loop-mediated isothermal amplification (LAMP) for malaria diagnosis in a field setting

We used the loop-mediated isothermal amplification (LAMP) method developed by our group for malaria diagnosis with genus-specific and species-specific primers for the four human malaria parasites at a field clinic in comparison with standard microscopy. Among 110 blood samples collected from the malaria clinic in Thailand, LAMP detected 59 of 60 samples positive by microscopy (sensitivity = 98.3%) and none of the 50 microscopy-negative samples (specificity = 100%). Negative predictive value (NPV) and positive predictive value (PPV) of LAMP were 98% and 100%, respectively. These results indicate that LAMP is an effective tool for malaria diagnosis at a field clinic in a field setting.     

环介导等温扩增技术检测蚊体内间日疟原虫子孢子的研究

目的建立一种简便、快速和高敏感度的蚊体内间日疟原虫检测的环介导等温扩增方法（LAMP）。方法针对间日疟原虫环子孢子蛋白（CSP）基因种属特异性保守区域6个位点设计2对引物,以感染性按蚊、阴性按蚊、恶性疟原虫及正常人血DNA为模板评价LAMP的特异性。将间日疟原虫CSP基因质粒DNA梯度稀释,并与阴性按蚊DNA按1.0μl加1.3×10^6、1.3×10^5、1.3×10^4、1.3×10^3、1.3×10^2、1.3×10^1、1.3×10^0拷贝混合后为模板进行LAMP反应,观察其检测敏感性;将感染性按蚊与阴性按蚊DNA作1：2、1：4、1：8、1：16、1：32、1：64、1：128、1：256稀释,然后以稀释样本为模板进行LAMP反应,观察批量检测的敏感性。再用此方法与镜检解剖、巢式PCR同时检测同批次人工感染的67只按蚊,评价其应用价值。结果此法检测感染性按蚊阳性,对照组均为阴性;检测不同比例稀释的间日疟原虫CSP基因质粒DNA与按蚊DNA混合物,最低可检测1.3×10^2拷贝的间日疟原虫CSP基因质粒DNA与按蚊DNA的混合物;检测不同比例感染按蚊与阴性按蚊DNA混合样本,最低可检测出在128个按蚊中有1个感染按蚊的混合样本;用此法检测67只同批次人工感染的按蚊,检出率为47.76%,解剖镜检检出率为25.37%（χ^2=7.24,P〈0.01）,巢式PCR检出率为40.30%（χ^2=0.73,P〉0.05）。以镜检解剖作为金标准,LAMP敏感性为100%,巢式PCR敏感性为100%。结论LAMP检测蚊体内间日疟原虫简便、快速、敏感性高,具有广阔的应用前景。     

Challenging loop-mediated isothermal amplification(LAMP) technique for molecular detection of Toxoplasma gondii

Objective:To compare analytical sensitivity and specificity of a newly described DNA amplification technique.LAMP and nested PCR assay targeting the RE and Bl genes for the detection of Toxoplasma gondii(T.gondii) DNA.Methods:The analytical sensitivity of LAMP and ncstcd-PCR was obtained against 10-fold serial dilutions of T.gondii DNA ranging from 1 ng to 0.01 fg.DNA samples of other parasites and human chromosomal DNA were used to determine the specificity of molecular assays.Results:After testing LAMP and nesled-PCR in duplicate,the detection limit of RE-LAMP.B1-LAMP,RE-nested PCR and B1-nested PCR assays was one fg.100 fg,1 pg and 10 pg of T.gondii DNA respectively.All the LAMP assays and nested PCRs were 100% specific.The RE-LAMP assay revealed the most sensitivity for the detection of T.gondii DNA.Conclusions:The obtained results demonstrate that the LAMP technique has a greater sensitivity for detection of T.gondii.Furthermore,these findings indicate that primers based on the RE are more suitable than those based on the B1 gene.However,the B1-LAMP assay has potential as a diagnostic tool for detection of T.gondii.     

结核分枝杆菌IS6110序列在石蜡组织中的检测、克隆与测序

目的：探讨套式-聚合酶链反应（Nested-PCR)检测石蜡组织中结核分枝杆菌的特异性和敏感性。方法：采用套式-PCR检测石蜡包埋组织中结核菌复合体特异插入序列IS6110，并对部分标本的PCR产物进行克隆和测序。结果：31例结核标本石蜡组织检出结核菌DNA共28例，套式-PCR的敏感度为90.3%,特异度为100%。阳性预测值为100%。随机选取两例PCR产物没是序结果与结核菌标准株H37Rv同源性分别为97%和95.3%。结论：套式-PCR检测常规石蜡包埋组织中结核菌IS6110序列具有特异性强和敏感性高的特点，可应用于临床诊断，尤其是对那些常规苏木精-伊红染色和抗酸染色无法确诊的病例更具意义。     

套式/多重PCR诊断疟疾的敏感性、特异性和稳定性初探

目的 提高标签引物?鄄套式/多重PCR诊断疟疾的敏感性、特异性与稳定性。 方法 用滤纸法采集非疟疾发热病人血样30份及其他传染病（感冒， 流感， 伤寒， 肝炎等）患者血样20份；抽取恶性疟和间日疟各1例患者血4 m1，进行系列稀释制备不同疟原虫含量的感染血样；健康者血样作对照。用热裂解法制备DNA模板，用线粒体细胞色素氧化酶基因作为靶基因，应用相关软件和网络资源（Primer Premier 5.0, PUBMED, NCBI-BLAST和Mfold服务器）设计和优化标签引物?鄄套式/多重PCR，并用于检测所采集制备的各种血样。 结果 间日疟与恶性疟患者血系列稀释为含 1 000、100、10和1个虫/μl后经标签引物?鄄套式/多重PCR检测，恶性疟和间日疟患者各稀释含虫血样分别在611 bp和255 bp出现1条带，能检测到原虫密度均为1个虫/μl血；非疟疾发热病人血样30份及其他传染病患者血样均为阴性；健康者血样未出现扩增条带，每种血样反复测试3次以上均获得同样结果。 结论 经优化的标签引物-套式/多重PCR在疟疾诊断中具有较高的敏感性、特异性和稳定性。     

A highly sensitive, nested polymerase chain reaction based method using simple dna extraction to detect malaria sporozoites in mosquitos

Dried Anopheles farauti mosquitos caught in Solomon Islands in 1990 were examined for malaria sporozoites by ELISA and nested polymerase chain reaction (PCR). Only heads and thoraces were used. Plasmodium genus-specific nested PCR amplifications were carried out on all samples. Of the 402 pools of mosquitos that were processed, 30 were positive for malaria. Nest 1 products of positive samples were subjected to further PCR amplifications with species-specific primers for P. falciparum and P. vivax. Twenty pools were positive for P. vivax by PCR while only 7 were positive by ELISA. For P. falciparum 2 pools were positive by both ELISA and PCR, and one of these was a pool which was positive for P. vivax by PCR and ELISA. Thus the sensitivity of PCR for P. vivax was 100% while the specificity was 96.7%. For P. falciparum the sensitivity and specificity were 100%. The PCR technique is highly sensitive and can be used on dried mosquitos which makes it a valuable tool for determining sporozoite rates of mosquitos, even in remote areas.     

环介导恒温扩增芯片法在下呼吸道感染病原体检测中的临床应用

目的分析环介导恒温扩增芯片法(LAMP)在下呼吸道感染病原体检测中的应用价值。 方法选择2018年1月至2018年9月空军军医大学第二附属医院收治的1 092例疑似下呼吸道感染患者,分析支气管肺泡灌洗液病原体检测结果,以细菌培养结果为金标准,评价LAMP检测8种常见下呼吸道感染病原体的灵敏度、特异度、阳性预测值、阴性预测值、阳性似然比、阴性似然比。 结果LAMP检测8种病原体的灵敏度差异较大,检测金黄色葡萄球菌灵敏度最高(100%),检测大肠埃希菌灵敏度最低(40%),检测8种病原体的特异度均高于94%。LAMP检测8种病原体的阳性预测值低于65%,阴性预测值高于98%,阳性似然比高于13,阴性似然比各病原体间差异较大,金黄色葡萄球菌阴性似然比最低(0.00),大肠埃希菌阴性似然比最高(0.60)。 结论LAMP在下呼吸道感染病原体检测中有较高的准确性,利于下呼吸道感染的早期诊断和精准治疗,具有临床意义。     

PCR technique for detecting Toxoplasma gondii in animal amniotic fluid

The goal of diagnosing congenital toxoplasmosis is early detection of maternofetal transmission, for early treatment to prevent unwanted sequelae. Polymerase chain reaction (PCR) is a method used recently for detecting toxoplasmosis during pregnancy. Amniotic fluid is a the clinical specimen used, since it provides a rapid, simple and safe method to obtain accurate results. The advantages of the PCR technique are high sensitivity, specificity and positive predictive value compared with other laboratory methods. To determine the sensitivity, specificity and lower detection limits in our laboratory, amplification of the B1 gene by nested PCR was performed on Toxoplasma gondii tachyzoites added to animal amniotic fluid samples. From 48 samples, our technique detected T. gondii in 30 out of 41 positive samples, and gave negative results for all the negative samples. The sensitivity for this nested PCR was 73%, the specificity was 100%, and the efficiency of the test was 77.1%. The nested PCR technique is recommended as a diagnostic method for detecting T. gondii in suspected congenital toxoplasmosis animals.