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The isothermal
ramification amplification assay is a feasible method for the detection of
Chlamydia trachomatis.
Detection of Chlamydia
trachomatis by isothermal ramification amplification method: a
feasibility study.
Zhang W, Cohenford M, Lentrichia B,
Isenberg HD, Simson E, Li H, Yi J, Zhang DY.
Journal of Clinical
Microbiology 2002;40:128-132.
Summary:
Question
Are the sensitivity and specificity
of the isothermal ramification (RAM) assay comparable to other molecular
methods for detection of C. trachomatis DNA in clinical specimens?
Design
This study describes a comparison of the results of the RAM assay with
those of a ligase chain reaction amplification assay (LCx) and a real-time
PCR assay for the detection of C. trachomatis DNA in residual
endocervical cell samples in PreservCyt cytological solution, selected
after analysis by LCx and PCR.
Participants
A group of 30 residual endocervical specimens in PreservCyt were selected
for the study. Fifteen specimens tested positive and 15 tested negative
for C. trachomatis DNA by LCx and PCR.
Description of Tests and Diagnostic
Standard
Cells were pelleted from the PreservCyt (Cytyc Corporation, Boxborough,
MA) solution by centrifugation and lysed in buffer specific for each
assay. The RAM assay, a novel, recently introduced, isothermal DNA
amplification method, consisted of steps including hybridization of the
amplification and capture probes to the target DNA, capture of the complex
onto magnetic beads (Dynal, Lake Success, NY), washing, ligation of the
amplification probe ends, and amplification of target DNA by primer
extension, strand displacement, and ramification reactions. The RAM
reaction products were digested with a restriction endonuclease and
analyzed on agarose gel. The real-time PCR assay was performed using a
LightCycler PCR System (Roche Molecular Biochemicals, Indianapolis, IN)
and the amplicon was detected with a molecular beacon. The ligase chain
reaction assay (LCx, Abbott Laboratories, Abbott Park, IL) was performed
according to the manufacturer's instructions.
Main Outcome Measures
The analytical sensitivity of the RAM assay and the detection of C.
trachomatis DNA in clinical PreservCyt samples compared to detection
by LCx and PCR assays were determined.
Main Results
The RAM assay detected the DNA from as few as 10 C. trachomatis
elementary bodies. All 15 clinical PreservCyt samples negative by LCx and
real-time PCR were also negative by RAM. Of the 15 samples that were
positive by LCx and PCR, 14 were positive by RAM.
Authors' Conclusions
The RAM assay, because of its relative
simplicity and isothermal amplification procedure, offers a viable
alternative to PCR and LCx for the detection of C. trachomatis DNA
from PreservCyt.
Source of funding: None given.
For correspondence: David Zhang,
Molecular Pathology Laboratory, Mount Sinai School of Medicine, Box 1122,
One Gustave Levy Place, New York, NY 10021. E-mail address: david.zhang@mountsinai.org.
expert
review
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