Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens

被引:46
作者
Cao, Qingqing [1 ]
Mahalanabis, Madhumita [2 ]
Chang, Jessie [2 ]
Carey, Brendan [2 ]
Hsieh, Christopher [2 ]
Stanley, Ahjegannie [2 ]
Odell, Christine A. [3 ,4 ]
Mitchell, Patricia [4 ]
Feldman, James [4 ]
Pollock, Nira R. [5 ]
Klapperich, Catherine M. [1 ,2 ]
机构
[1] Boston Univ, Dept Mech Engn, Boston, MA 02215 USA
[2] Boston Univ, Dept Biomed Engn, Boston, MA 02215 USA
[3] Boston Med Ctr, Dept Pediat, Div Pediat Emergency Med, Boston, MA USA
[4] Boston Univ, Sch Med, Boston, MA USA
[5] Harvard Med Sch, Beth Israel Deaconess Med Ctr, Div Infect Dis, Boston, MA USA
来源
PLOS ONE | 2012年 / 7卷 / 03期
基金
美国国家卫生研究院;
关键词
NUCLEIC-ACID AMPLIFICATION; SYNCYTIAL VIRUS; CHEMICAL-ANALYSIS; GENETIC-ANALYSIS; H1N1; VIRUS; PCR; SENSITIVITY; PERFORMANCE; SYSTEM; INFECTION;
D O I
10.1371/journal.pone.0033176
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
A rapid, low cost, accurate point-of-care (POC) device to detect influenza virus is needed for effective treatment and control of both seasonal and pandemic strains. We developed a single-use microfluidic chip that integrates solid phase extraction (SPE) and molecular amplification via a reverse transcription polymerase chain reaction (RT-PCR) to amplify influenza virus type A RNA. We demonstrated the ability of the chip to amplify influenza A RNA in human nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens collected at two clinical sites from 2008-2010. The microfluidic test was dramatically more sensitive than two currently used rapid immunoassays and had high specificity that was essentially equivalent to the rapid assays and direct fluorescent antigen (DFA) testing. We report 96% (CI 89%, 99%) sensitivity and 100% (CI 95%, 100%) specificity compared to conventional (bench top) RT-PCR based on the testing of n = 146 specimens (positive predictive value = 100%(CI 94%, 100%) and negative predictive value = 96%(CI 88%, 98%)). These results compare well with DFA performed on samples taken during the same time period (98% (CI 91%, 100%) sensitivity and 96%(CI 86%, 99%) specificity compared to our gold standard testing). Rapid immunoassay tests on samples taken during the enrollment period were less reliable (49%(CI 38%, 61%) sensitivity and 98%(CI 98%, 100%) specificity). The microfluidic test extracted and amplified influenza A RNA directly from clinical specimens with viral loads down to 10(3) copies/ml in 3 h or less. The new test represents a major improvement over viral culture in terms of turn around time, over rapid immunoassay tests in terms of sensitivity, and over bench top RT-PCR and DFA in terms of ease of use and portability.
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页数:11
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