Rapid and sensitive detection ofChlamydia trachomatisusing a ligatable binary RNA probe and Qβ replicase

Stefano, James E.; Genovese, Luigi; An, Qing; Lü, Ling; McCarty, Jack C.; Du, Yuguo; Stefano, Kelly; Burg, Josef; King, Walter E.; Lane, David

doi:10.1006/mcpr.1997.0135

Cited by 6 publications

(3 citation statements)

References 31 publications

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“…A magnetic capture technique was developed to isolate Ehrlichia/Anaplasma 16S rRNA from in vitro cultures or blood by following a strategy similar to the one reported for Chlamydia trachomatis, 26 but with several modifications ( Figure 3). An Ehrlichia and Anaplasma genera-specific capture primer was designed from the complementary sequence of 16S rRNA that is conserved in all known Ehrlichia/Anaplasma species.…”

Section: S Rrnamentioning

confidence: 99%

Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Sirigireddy

Ganta

2005

The Journal of Molecular Diagnostics

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Tick-borne infections are responsible for many emerging diseases in humans and several vertebrates. These include human infections with Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Because single or co-infections can result from tick bites, the availability of a rapid, multiplex molecular test will be valuable for timely diagnosis and treatment. Here, we describe a multiplex molecular test that can detect single or co-infections with up to five Ehrlichia and Anaplasma species. The test protocol includes the magnetic capture-based purification of 16S ribosomal RNA, its enrichment, and specific-pathogen(s) detection by real-time reverse transcriptase-polymerase chain reaction. We also report a unique cloning strategy to develop positive controls in the absence of a pathogen's genomic DNA. The test was assessed by examining blood samples from dogs suspected to be positive for ehrlichiosis. The dog was chosen as the model system because it is susceptible to acquire infections with up to five pathogens of the genera Ehrlichia and Anaplasma. The test identified single infections in the canine host with E. chaffeensis, E. canis, E. ewingii, A. phagocytophilum, and A. platys and co-infection with E. canis and A. platys. The multipathogen detection and novel positive control development procedures described here will be valuable in monitoring infections in people, other vertebrates, and ticks. (J Mol Diagn 2005, 7:308 -316) Several rickettsial agents of the family Anaplasmataceae cause severe, tick-borne pathogen infections in a wide range of vertebrate host species.

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Section: S Rrnamentioning

confidence: 99%

Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Sirigireddy

Ganta

2005

The Journal of Molecular Diagnostics

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“…Recent investigations have focused on splitting the midivariant RNA detector probe into two pieces which can replicate only when ligated following hybridization with specific target. Using this "smart probe" approach, 10 2 target molecules of Chlamydia or human immunodeficiency virus RNA have been detected in model systems by using only one round of RTC (42,47). These initial results with a simplified assay are very encouraging and should stimulate further feasibility studies on adapting Q-Beta replicase amplification to existing instruments.…”

Section: Discussionmentioning

confidence: 97%

Performance of an automated Q-beta replicase amplification assay for Mycobacterium tuberculosis in a clinical trial

Smith¹,

Radcliffe²,

Rigby³

et al. 1997

J Clin Microbiol

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We present data from a clinical trial study in which an automated version (Galileo) of a previously described Q-Beta replicase-amplified probe assay (J. S. Shah et al., J. Clin. Microbiol. 33:1435-1441, 1995) was used for the direct detection of Mycobacterium tuberculosis complex in sputum. The assay was designed to target specific regions of 23S rRNA found in M. tuberculosis, Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium microti and had a sensitivity ranging from approximately <10 to 300 CFU. The assay was tested for cross-hybridization by using large numbers (e.g., 10 5 to 10 10 CFU/assay) of 133 other organisms commonly found in respiratory tract samples, including non-M. tuberculosis Mycobacterium spp., other bacteria, fungi, and viruses. All of these competitors tested negative by the assay. Automated assay results for 780 respiratory tract samples (sputum or bronchoalveolar lavage specimens) collected and tested at three trial sites in the United States) were compared with the results of culture and acid-fast microscopy. Aliquots of conventionally digested and decontaminated sputum pellets were heated at 100°C and mechanically disrupted prior to hybridization and background reduction, amplification, and detection in a closed disposable test pack. Pertinent elements of individual patient histories relating to tuberculosis exposure, previous active disease, antituberculosis therapy status, etc., were considered in the resolution of discrepant results for 48 (assay false-positive) samples. Seventy-one of 90 (78.9%) culture-positive samples were positive when tested in the Galileo assay, while 7% of culture-negative samples were assay positive, corresponding to a sensitivity of 79% and a specificity of 93%. Following resolution of discrepant results by chart review, the sensitivity and specificity for the Q-Beta replicase amplification assay with the Galileo analyzer were 84 and 97%, respectively. A total of 69.2% of smear-negative (culture positive) samples were detected by the assay. Ten test packs at a time were automatically processed by the Galileo analyzer without operator intervention following loading of samples. The first result was reported in approximately 3 h, and the last result was available in 6.5 h. To our knowledge, this is the first report of a clinical study with a fully automated amplification probe hybridization assay for the detection of pathogens directly from a clinical specimen.

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“…These commercial tests manufactured by GenProbe (San Diego, CA) were initially aimed at direct ribosomal RNA detection, the second generation of tests was also able to generate limited ribosomal RNA amplification (Verkooyen et al, 2003). Ultimately, various amplification mediated tests were developed based on technologies such as bacteriophage Qb RNA polymerase mediated amplification (Stefano et al, 1997), the ligase chain reaction or LCR (Blocker et al, 2002), and, of course, the PCR (see Verkooyen et al, 2003 and references therein). These tests all underwent extensive comparisons with the culture-based assay and among each other, and ultimately the PCR tests prevailed, although the LCR tests demonstrated adequate sensitivity and specificity (Pannekoek et al, 2003).…”

Section: Commercial Test Systems For Chlamydia Trachomatis Diagnosismentioning

confidence: 99%

Molecular Diagnostics and Comparative Genomics in Clinical Microbiology

Belkum

2010

Molecular Diagnostics

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Rapid and sensitive detection ofChlamydia trachomatisusing a ligatable binary RNA probe and Qβ replicase

Cited by 6 publications

References 31 publications

Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Performance of an automated Q-beta replicase amplification assay for Mycobacterium tuberculosis in a clinical trial

Molecular Diagnostics and Comparative Genomics in Clinical Microbiology

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