Rapid and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus by Rolling Circle Amplification

Abstract

The severe acute respiratory syndrome (SARS) epidemic of 2003 was responsible for 774 deaths and caused significant economic damage worldwide. Since July 2003, a number of SARS cases have occurred in China, raising the possibility of future epidemics. We describe here a rapid, sensitive, and highly efficient assay for the detection of SARS coronavirus (SARS-CoV) in cultured material and a small number (n ‫؍‬ 7) of clinical samples. Using rolling circle amplification (RCA), we were able to achieve sensitive detection levels of SARS-CoV RNA in both solid and liquid phases. The main advantage of RCA is that it can be performed under isothermal conditions with minimal reagents and avoids the generation of false-positive results, a problem that is frequently encountered in PCR-based assays. Furthermore, the RCA technology provides a faster, more sensitive, and economical option to currently available PCR-based methods.Severe acute respiratory syndrome (SARS) is an emerging disease caused by the novel SARS coronavirus (SARS-CoV) (2,4,5,14). By the end of the SARS epidemic in July 2003, a total of 8,096 SARS cases had been reported from 30 countries, with 774 deaths. Whether future outbreaks of SARS will occur is unknown at present. However, given the recent SARS cases in southern China arising from an unknown source and a number of laboratory-related infections (12), it is important to be prepared for such a possibility. In the absence of a SARSCoV vaccine or antiviral drugs, the use of strict infection control policies and early diagnosis with rapid, sensitive, and highly specific laboratory methods are essential for the early management of SARS-CoV infection.Apart from epidemiological linkages, the clinical and radiographic features of the disease are not SARS specific, identifying a need for specific laboratory tests that can confirm SARS-CoV infection early in the course of the illness. Detection of SARS-CoV-specific antibodies is a sensitive and specific but is not possible at clinical presentation (6,14).Detection of SARS-CoV by reverse transcription-PCR (RT-PCR) in clinical specimens allows diagnosis in the early stage of the disease. However, in contrast to many other acute respiratory infections, only low levels of SARS-CoV are thought to be present during the early symptomatic phase of infection. On the basis of the results of first-generation RT-PCR assays, SARS-CoV RNA can be detected with a sensitivity of only ca. 30 to 50% in a single respiratory specimen. A higher sensitivity can be achieved if serial samples are collected, particularly during the second week of illness when maximal virus shedding occurs (13,14). The type of clinical sample (e.g., nasopharyngeal aspirate, throat swabs, stool samples, urine, etc.) also affects the sensitivity of .Recently, the utility of circularizable oligonucleotides, or "padlock probes," has been demonstrated for the detection of target nucleic acid sequences; this approach shows greater sensitivity than conventional PCR (3,8,16). Upon hybridization to a target DNA or...

Hyperbranched rolling circle amplification as a rapid and sensitive method for species identification within the Cryptococcus species complex

“…1) [21]. This technology has been used for SNP genotyping in human populations [18][19][20], as well as for the detection of human pathogenic viruses [22] and bacteria [23]. In this study, we investigate the use of padlock probes used in combination with HRCA and real-time PCR for highthroughput rapid identification and differentiation of isolates of the Cryptococcus species complex.…”

Practical Method for Detection and Identification of Candida, Aspergillus , and Scedosporium spp. by Use of Rolling-Circle Amplification

“…As a result, new priming sites for the first primer (primer 1) are generated. The two primers thus function to generate a self-propagating pattern of DNA fragment release events (20).…”

“…In particular, the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the fungal ribosomal DNA gene complex have shown promise as targets for species identification in a variety of formats including multiplex and/or real-time PCR assays (9, 16), DNA sequence analysis (1, 2, 12), and probe-based techniques (5, 7). The latter range from Southern blotting (5, 7) and reverse line blot (RLB) hybridization methods (23) to sophisticated microarray formats (10,11,17).Recently, the utility of circularizable oligonucleotide (padlock) probes has been demonstrated for the detection of target nucleic acid sequences, including nucleotide polymorphisms that differ by only a few base pairs, with high sensitivity (4,13,20). Such probes comprise two sequences complementary to the 5Ј and 3Ј termini of the target sequence joined by a linker region (Fig.…”

“…Recently, the utility of circularizable oligonucleotide (padlock) probes has been demonstrated for the detection of target nucleic acid sequences, including nucleotide polymorphisms that differ by only a few base pairs, with high sensitivity (4,13,20). Such probes comprise two sequences complementary to the 5Ј and 3Ј termini of the target sequence joined by a linker region (Fig.…”

“…RCA reactions of the circularized probes were then performed according to previously described experimental conditions (18,20). Probe signals were amplified by incubation at 65°C for 30 min, and accumulation of doublestranded DNA products was monitored using a Corbett Ro-torGene 6000 machine (Corbett Research, Mortlake, Australia); probe signals were also visualized on a 1.5% agarose gel to verify the specificity of probe-template binding.…”