Detection of rRNA from four respiratory pathogens using an automated Qβ replicase assay

Stone, Benjamin B.; Cohen, Stanley; Breton, Gary L.; Nietupski, Raymond M.; Pelletier, Dale A.; Fiandaca, Mark J.; Moe, James G.; Smith, James H.; Shah, Jyotsna; Weisburg, William G.

doi:10.1006/mcpr.1996.0049

Cited by 13 publications

(6 citation statements)

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“…Further testing at clinical trial sites has been undertaken to verify the practical application of the prototype instrument platform and the performance and robustness of the basic Q-Beta replicase amplification technology and is described in an accompanying report (20). Finally, we have recently described model assays for several other organisms using Q-Beta replicase amplification and the Galileo platform (6,22).…”

Section: Discussionmentioning

confidence: 99%

Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay

Smith¹,

Buxton²,

Cahill³

et al. 1997

J Clin Microbiol

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We have adapted an assay for the direct detection of Mycobacterium tuberculosis using a prototype automated instrument platform in which probes are amplified with Q-beta replicase. The assay was based on amplification of specific detector probe following four cycles of background reduction (reversible target capture) in a closed disposable pack. The assay signal was the time required for fluorescence to exceed background levels (response time [RT]). RT was inversely related to the number of M. tuberculosis rRNA target molecules in the sample. Equivalent signals and noises were observed in assays containing either sputum or buffer. All mock samples containing >10 CFU of M. tuberculosis responded in the assay (average RT, 13.91 min), while most (83%) samples containing as many as 10 7 CFU of Mycobacterium avium gave no response during a 25-min amplification reaction. The samples containing M. avium which did respond had an average RT of 17.04 min. Seventy-five percent (167 of 223) of samples containing no target gave no responses; the remaining 25% had an average RT of 15.53 min. Eighty-three frozen sputum samples were tested to develop a candidate cutoff RT for the assay prior to more extensive clinical testing. After resolution of discrepant results and with a 14-min RT cutoff, 30 of 38 M. tuberculosis-positive samples were positive by the assay; 1 of 45 negative samples responded within 14 min. Assay sensitivity, specificity, and positive and negatives predictive values in this pilot study were 79, 98, 97, and 85%, respectively.

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Section: Discussionmentioning

confidence: 99%

Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay

Smith¹,

Buxton²,

Cahill³

et al. 1997

J Clin Microbiol

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“…Direct capture of target gene sequences by hybridization with oligonucleotide probes was performed on supernatants. Briefly, 10 mL polyvinylpolypyrrolidone-treated supernatant was denatured in 2.4 M guanidine isothiocyanate (Sigma, St Louis, MO) at 90°C for 10 min; 500 μ g Sera-Mag carboxylate modified beads (ThermoFisher Scientific, Waltham, MA) functionalized with each oligonucleotide capture probe 26 were subsequently added to denatured stool supernatant and incubated at room temperature for 1 hour. Sera-Mag beads were collected on a magnetic rack, and washed 3 times using 1× MOPS washing buffer (10 mM MOPS, 150 mM NaCl, pH 7.5), and then eluted out in 50 μ L nuclease-free water with 20 ng/ μ L transfer RNA (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Next-Generation Stool DNA Test Accurately Detects Colorectal Cancer and Large Adenomas

et al. 2012

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BACKGROUND & AIMS Technical advances have led to stool DNA (sDNA) tests that might accurately detect neoplasms on both sides of the colorectum. We assessed colorectal neoplasm detection by a next-generation sDNA test and effects of covariates on test performance. METHODS We performed a blinded, multicenter, case-control study using archived stool samples collected in preservative buffer from 252 patients with colorectal cancer (CRC), 133 with adenomas ≥1 cm, and 293 individuals with normal colonoscopy results (controls); two-thirds were randomly assigned to a training set and one-third to a test set. The sDNA test detects 4 methylated genes, a mutant form of KRAS, and the α-actin gene (as a reference value) using quantitative, allele-specific, real-time target and signal amplification; it also quantifies hemoglobin. We used a logistical model to analyze data. RESULTS The sDNA test identified 85% of patients with CRC and 54% of patients with adenomas ≥1 cm with 90% specificity. The test had a high rate of detection for all nonmetastatic stages of CRC (aggregate 87% detection rate for CRC stages I–III). Detection rates increased with adenoma size: 54% ≥1 cm, 63% >1 cm, 77% >2 cm, 86% >3 cm, and 92% >4 cm (P < .0001). Based on receiver operating characteristic analysis, the rate of CRC detection was slightly greater for the training than the test set (P = .04), whereas the rate of adenoma detection was comparable between sets. Sensitivities for detection of CRC and adenoma did not differ with lesion site. CONCLUSIONS Early-stage CRC and large adenomas can be detected throughout the colorectum and with high levels of accuracy by the sDNA test. Neoplasm size, but not anatomical site, affected detection rates. Further studies are needed to validate the findings in a larger population and optimize the sDNA test.

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“…Besides PCR, alternative amplification techniques, such as NASBA (55), transcription-mediated amplification, ligase chain reaction, Q␤ replicase amplification (63), and strand displacement amplification, have been developed. These techniques may be useful alternatives to PCR, but so far, few studies on using these techniques to detect M. pneumoniae in respiratory specimens have been published, except for NASBA.…”

Section: Technical Aspectsmentioning

confidence: 99%

“…Later on, NASBA in combination with ELGA and electrochemiluminescence detection was used to detect M. pneumoniae RNA in nucleic acid extracts from respiratory specimens (47). The Q␤ replicase assay was applied to detect synthetic M. pneumoniae 16S rRNA transcripts and seems to be less sensitive than PCR (63).…”

Section: Technical Aspectsmentioning

confidence: 99%

Molecular Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infections

et al. 2003

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2Mycoplasma pneumoniae is responsible for 10 to 20% of the cases of community-acquired pneumonia and has been associated with acute exacerbations of asthma (22). M. pneumoniae is also implicated in mild acute respiratory infections, such as sore throat, pharyngitis, rhinitis, and tracheobronchitis (2).Correct diagnosis of M. pneumoniae infections is important to allow the appropriate antibiotic treatment of patients, since it is impossible to identify a M. pneumoniae infection solely on the basis of clinical signs and symptoms. It should decrease inappropriate use of antibiotics, influence the patient outcome by reduction of morbidity and mortality, and improve our knowledge of the prevalence of the causes of so-called atypical pneumonia.Conventional assays for the detection of M. pneumoniae have their limitations, resulting in the need for more accurate diagnostic methods. Culture is time-consuming and relatively insensitive, because M. pneumoniae grows slowly in vitro, requiring 2 to 5 weeks for colonies to become visible. Serological methods, particularly the complement fixation (CF) test, are most widely used. The sensitivity of these assays depends on whether the first serum sample is collected early or late after the onset of disease and on the availability of paired serum samples collected with an interval of 2 to 3 weeks. Immunoglobulin M (IgM) assays which are more sensitive than the CF test have been developed, but the IgM response may be nonspecific (61) or absent, particularly in adults (70). Hybridization with DNA probes has also been proposed as a rapid and specific procedure to replace culture, but it lacks sensitivity (35).Nucleic acid amplification techniques (NAATs) have the potential to produce rapid, sensitive, and specific results, allowing early appropriate antibiotic therapy.In the absence of a reference method, the so-called "gold standard" for the diagnosis of an M. pneumoniae infection, either an expanded gold standard or the technique of latent class analysis (LCA) should be applied to calculate the sensitivity and specificity of the available diagnostic tests. The technique of LCA can be used if at least three independent techniques can be compared. Thus far, only a few PCR tests and a limited number of studies applying culture, serology, and several NAATs targeting different genes to detect M. pneumoniae have been adequately evaluated.Since NAATs targeting DNA can detect both viable and nonviable organisms, detecting RNA by reverse transcriptase PCR (RT-PCR) or nucleic acid sequence-based amplification (NASBA) may be a useful method to identify productive M. pneumoniae infections.The possible long-term carrier state of M. pneumoniae in the respiratory tract may hinder the evaluation of different diagnostic tests for the diagnosis of acute infections.An overview of the peer-reviewed literature on the use of NAATs to detect M. pneumoniae since 1989 is given. Search combinations were M. pneumoniae and PCR, M. pneumoniae and diagnosis, and M. pneumoniae and amplification. This minireview...

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Detection of rRNA from four respiratory pathogens using an automated Qβ replicase assay

Cited by 13 publications

References 0 publications

Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay

Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay

Next-Generation Stool DNA Test Accurately Detects Colorectal Cancer and Large Adenomas

Molecular Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infections

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