Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus

Abstract

The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 10(2) PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63 degrees C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries.

FIG. 1.

Oligonucleotide primers used for RT-LAMP amplification of SARS-CoV (GenBank accession number NC-004718). The underlined letters indicate the sequences of primers.

FIG. 2.

Agarose gel electrophoresis and restriction analysis of RT-LAMP products of the Rep gene of SARS-CoV on a 3% agarose gel. Lane M, 100-bp DNA ladder (Sigma Genosys, Hokkaido, Japan); lane 1, RT-LAMP product of SARS-CoV digested with HpaI (135 bp); lane 2, RT-LAMP products of SARS-CoV; lane 3, RT-LAMP without target RNA.

FIG. 3.

Real-time kinetics of RT-LAMP amplification of SARS-CoV RNA, as monitored by real-time measurement of turbidity on a Loopamp real-time turbidimeter (LA-200; Teramecs).

FIG. 4.

Comparative sensitivity of RT-LAMP and RT-PCR for detection of SARS-CoV. The amplification by RT-LAMP (A) showed ladder-like pattern, whereas the RT-PCR (B) showed 195-bp amplification. (A) Sensitivity of SARS RT-LAMP assay as monitored by real-time measurement of turbidity (LA-200; Teramecs). Shown from left to right are the curves of decreasing concentrations of virus from 10,000 to 0.00001 PFU. The detection limit for the assay was 0.01 PFU. (B) Sensitivity of RT-PCR for the detection of SARS-CoV as observed by agarose gel analysis with a detection limit of 1 PFU.

FIG. 5.

Quantitative determination of virus concentration in clinical samples employing a standard curve of the SARS RT-LAMP assay. (A) Standard curve generated for SARS RT-LAMP assay by plotting T_p with regard to various concentrations of virus in PFU. (B) Determination of virus titer in clinical samples derived from the standard curve based on their T_p values.