Published online 2011 Jan 11. doi: 10.1371/journal.pntd.0000931
PMCID: PMC3019106
International Study to Evaluate PCR Methods for Detection of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients
对美洲锥虫病人血液样品中锥虫DNA的PCR检测方法的国际研究评估
以下是参与研究人员：
Alejandro G. Schijman,1,* Margarita Bisio,1 Liliana Orellana,2 Mariela Sued,2 Tomás Duffy,1 Ana M. Mejia Jaramillo,3Carolina Cura,1 Frederic Auter,4 Vincent Veron,5 Yvonne Qvarnstrom,6 Stijn Deborggraeve,7 Gisely Hijar,8 Inés Zulantay,9 Raúl Horacio Lucero,10 Elsa Velazquez,11 Tatiana Tellez,12 Zunilda Sanchez Leon,13 Lucia Galvão,14Debbie Nolder,15 María Monje Rumi,16 José E. Levi,17 Juan D. Ramirez,18 Pilar Zorrilla,19 María Flores,20 Maria I. Jercic,21 Gladys Crisante,22 Néstor Añez,22 Ana M. De Castro,23 Clara I. Gonzalez,24 Karla Acosta Viana,25 Pedro Yachelini,26 Faustino Torrico,12 Carlos Robello,1
9 Patricio Diosque,16 Omar Triana Chavez,3 Christine Aznar,5Graciela Russomando,13 Philippe Büscher,7 Azzedine Assal,4 Felipe Guhl,18 Sergio Sosa Estani,27 Alexandre DaSilva,6 Constança Britto,28 Alejandro Luquetti,29 and Janis Ladzins30
Ana Rodriguez, Editor [Ana Rodriguez,编辑;]
Abstract [摘要]
Background [背景]
A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.
自锥虫病发现后的一个世纪以来，查加斯锥虫病仍然代表一种易被忽视的主要的热带威胁。精准的诊断学工具同时也作为上寄生虫学反应的替代标志，在寄生虫研究领域是优先考
虑的。这项研究通过外部的质量评估方法来评价PCR检测方法在对克氏锥虫的DNA进行检测时的效果。
Methodology/Findings [ 生物学方法/检测发现的情况]
An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests d
etected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four method
s were further evaluated at the coordinating laboratory in human blood samples, confirming the performance obtained by the participating laboratories.
来自16个国家的专业的实验室开展了一项国际合作研究。目前对已提纯的克氏锥虫DNA进行连续稀释的生物研究策略受到了挑战，这些DNA来自于克氏锥虫DNA的离散分型单元(DTU)I, IV 和VI （set A），已经加入寄生虫细胞的人类血液（set B）和取自南美洲锥状地区国家的32个血清反应阳性的和10个阴性病人的Guanidine Hidrochloride-EDTA 血样（set C）.已报道的set A的PCR检测有48例，set B和set C共有44例报道。28个靶向微环DNA（DNA），13个卫星DNA（Sat-DNA）和剩下的低复制序列的DNA.在set A中，商业性的硕士混合和Sat-DNA RealTime PCR 显示更为明确，但是kDNA的PCR检测比DTU I 型DNA更为敏感。在set B中，与溶剂萃取协议相比，商业DNA提取试剂盒表现更好的特异性。Sat-DNA 以每毫升0.05-0.5寄生虫的灵敏度，使其PCR检测具有更高的特异性，而特定的k-DNA每毫升5.10−3帕尔才检测出。 16个明确清晰的检测在setA和set B上都有较好地体现（10 fg/µl of 所有库存DNA , 5 par/mL 在已经入寄生虫细胞的血样中）。 通过分析Set A和Set B相当多地变化的16例检测中，检测setC中获得了其敏感度，特异性和精确度的中值。除此之外，4种方法描述在全部的三种血样中最好的表现参数，描述为至少每个DNA库存10 fg/µl ，0.5
par/mL 以及 83.3–94.4%,之间的灵敏度， 85–95%之间的特异性，86.8–89.5% 之间的精确度以及0.7-0.8的kappa指数， 与16个表现好的检测的63–69%, 100%, 71.4–76.2% and 0.4–0.5相比，分别比较血清学诊断。Sybr-Green 所遵从的使用溶剂萃取的LbD2方法基于Real Time PCR对Sat-DNA的定向；
LbD3使用传统PCR对Sat-A进行定向；第三种方法(LbF1) 使用基于DNA萃取的玻璃纤维柱，为探针Real Time PCR技术用来针对Sat-DNA（克氏1/克氏2/克氏3探针法）。第四种方法（LbQ）就是使用溶剂DNA萃取，然后用传统的热启动PCR针对k-DNA（引物对数121/122）.这四种方法在协作实验室更深入的评估人类血样，确定了参与实验室所获得的性能。
Conclusion/Significance 结论/重大意义
This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.这项研究是在PCR程序对血样中克氏锥虫检测所迈出的至关重要的第一步。
This article from NCBI is cited by me.
本文选自NCBI.英文链接：bi.v/pmc/articles/PMC3019106/
1.Chagas' disease 南美锥虫病 ; 恰加斯氏病 ; 南美洲锥虫病 ; 恰加斯病
2.Trypanosome cruzi 克氏锥虫 ; 弓形虫
3.Tropical geometry 热带几何
3.accurate model 准确模型 ; 精确模型 ; 精确模型 ; 精确的模型ntd
4.quality diagnostics 质量诊断 ; 质量诊断学
5.surrogate products 替代产品 ; 更换产品
6.Parasitological Testing 寄生虫学检验
7.external environment 外部环境 ; 外环境 ; 外在环境 ; 外界环境
8.Tropical geometry 热带几何
9.accurate model 准确模型 ; 精确模型 ; 精确模型 ; 精确的模型
10.quality diagnostics 质量诊断 ; 质量诊断学
11.surrogate products 替代产品 ; 更换产品
12.Parasitological Testing 寄生虫学检验
13.external environment 外部环境 ; 外环境 ; 外在环境 ; 外界环境
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