PMID- 21995644
OWN - NLM
STAT- MEDLINE
DCOM- 20120227
LR  - 20240314
IS  - 1520-5126 (Electronic)
IS  - 0002-7863 (Print)
IS  - 0002-7863 (Linking)
VI  - 133
IP  - 44
DP  - 2011 Nov 9
TI  - Multiplexed quantification of nucleic acids with large dynamic range using 
      multivolume digital RT-PCR on a rotational SlipChip tested with HIV and hepatitis 
      C viral load.
PG  - 17705-12
LID - 10.1021/ja2060116 [doi]
AB  - In this paper, we are working toward a problem of great importance to global 
      health: determination of viral HIV and hepatitis C (HCV) loads under 
      point-of-care and resource limited settings. While antiretroviral treatments are 
      becoming widely available, viral load must be evaluated at regular intervals to 
      prevent the spread of drug resistance and requires a quantitative measurement of 
      RNA concentration over a wide dynamic range (from 50 up to 10(6) molecules/mL for 
      HIV and up to 10(8) molecules/mL for HCV). "Digital" single molecule measurements 
      are attractive for quantification, but the dynamic range of such systems is 
      typically limited or requires excessive numbers of compartments. Here we designed 
      and tested two microfluidic rotational SlipChips to perform multivolume digital 
      RT-PCR (MV digital RT-PCR) experiments with large and tunable dynamic range. 
      These designs were characterized using synthetic control RNA and validated with 
      HIV viral RNA and HCV control viral RNA. The first design contained 160 wells of 
      each of four volumes (125 nL, 25 nL, 5 nL, and 1 nL) to achieve a dynamic range 
      of 5.2 x 10(2) to 4.0 x 10(6) molecules/mL at 3-fold resolution. The second 
      design tested the flexibility of this approach, and further expanded it to allow 
      for multiplexing while maintaining a large dynamic range by adding additional 
      wells with volumes of 0.2 nL and 625 nL and dividing the SlipChip into five 
      regions to analyze five samples each at a dynamic range of 1.8 x 10(3) to 1.2 x 
      10(7) molecules/mL at 3-fold resolution. No evidence of cross-contamination was 
      observed. The multiplexed SlipChip can be used to analyze a single sample at a 
      dynamic range of 1.7 x 10(2) to 2.0 x 10(7) molecules/mL at 3-fold resolution 
      with limit of detection of 40 molecules/mL. HIV viral RNA purified from clinical 
      samples were tested on the SlipChip, and viral load results were self-consistent 
      and in good agreement with results determined using the Roche COBAS 
      AmpliPrep/COBAS TaqMan HIV-1 Test. With further validation, this SlipChip should 
      become useful to precisely quantify viral HIV and HCV RNA for high-performance 
      diagnostics in resource-limited settings. These microfluidic designs should also 
      be valuable for other diagnostic and research applications, including detecting 
      rare cells and rare mutations, prenatal diagnostics, monitoring residual disease, 
      and quantifying copy number variation and gene expression patterns. The theory 
      for the design and analysis of multivolume digital PCR experiments is presented 
      in other work by Kreutz et al.
FAU - Shen, Feng
AU  - Shen F
AD  - Department of Chemistry and Institute for Biophysical Dynamics, The University of 
      Chicago, 929 East 57th Street, Chicago, Illinois 60637, United States.
FAU - Sun, Bing
AU  - Sun B
FAU - Kreutz, Jason E
AU  - Kreutz JE
FAU - Davydova, Elena K
AU  - Davydova EK
FAU - Du, Wenbin
AU  - Du W
FAU - Reddy, Poluru L
AU  - Reddy PL
FAU - Joseph, Loren J
AU  - Joseph LJ
FAU - Ismagilov, Rustem F
AU  - Ismagilov RF
LA  - eng
GR  - R01 EB012946-01/EB/NIBIB NIH HHS/United States
GR  - 1R01 EB012946/EB/NIBIB NIH HHS/United States
GR  - 1 DP1 OD003584/OD/NIH HHS/United States
GR  - R01 EB012946/EB/NIBIB NIH HHS/United States
GR  - DP1 OD003584/OD/NIH HHS/United States
GR  - DP1 OD003584-01/OD/NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20111013
PL  - United States
TA  - J Am Chem Soc
JT  - Journal of the American Chemical Society
JID - 7503056
RN  - 0 (RNA, Viral)
SB  - IM
MH  - HIV/*chemistry/genetics
MH  - Hepacivirus/*chemistry/genetics
MH  - *Microfluidic Analytical Techniques
MH  - RNA, Viral/*analysis/genetics
MH  - *Reverse Transcriptase Polymerase Chain Reaction
MH  - Rotation
MH  - *Viral Load
PMC - PMC3216675
MID - NIHMS332187
EDAT- 2011/10/15 06:00
MHDA- 2012/03/01 06:00
PMCR- 2012/11/09
CRDT- 2011/10/15 06:00
PHST- 2011/10/15 06:00 [entrez]
PHST- 2011/10/15 06:00 [pubmed]
PHST- 2012/03/01 06:00 [medline]
PHST- 2012/11/09 00:00 [pmc-release]
AID - 10.1021/ja2060116 [doi]
PST - ppublish
SO  - J Am Chem Soc. 2011 Nov 9;133(44):17705-12. doi: 10.1021/ja2060116. Epub 2011 Oct 
      13.