Browsing by Subject "Nucleic Acid Amplification Techniques"

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    A paired-end sequencing strategy to map the complex landscape of transcription initiation.
    (Nature methods, 2010-07) Ni, Ting; Corcoran, David L; Rach, Elizabeth A; Song, Shen; Spana, Eric P; Gao, Yuan; Ohler, Uwe; Zhu, Jun
    Recent studies using high-throughput sequencing protocols have uncovered the complexity of mammalian transcription by RNA polymerase II, helping to define several initiation patterns in which transcription start sites (TSSs) cluster in both narrow and broad genomic windows. Here we describe a paired-end sequencing strategy, which enables more robust mapping and characterization of capped transcripts. We used this strategy to explore the transcription initiation landscape in the Drosophila melanogaster embryo. Extending the previous findings in mammals, we found that fly promoters exhibited distinct initiation patterns, which were linked to specific promoter sequence motifs. Furthermore, we identified many 5' capped transcripts originating from coding exons; our analyses support that they are unlikely the result of alternative TSSs, but rather the product of post-transcriptional modifications. We demonstrated paired-end TSS analysis to be a powerful method to uncover the transcriptional complexity of eukaryotic genomes.
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    An enhanced isothermal amplification assay for viral detection.
    (Nature communications, 2020-11) Qian, Jason; Boswell, Sarah A; Chidley, Christopher; Lu, Zhi-Xiang; Pettit, Mary E; Gaudio, Benjamin L; Fajnzylber, Jesse M; Ingram, Ryan T; Ward, Rebecca H; Li, Jonathan Z; Springer, Michael
    Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.
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    Simultaneous Evaluation of Diagnostic Assays for Pharyngeal and Rectal Neisseria gonorrhoeae and Chlamydia trachomatis Using a Master Protocol.
    (Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 2020-12) Doernberg, Sarah B; Komarow, Lauren; Tran, Thuy Tien T; Sund, Zoe; Pandori, Mark W; Jensen, David; Tsalik, Ephraim L; Deal, Carolyn D; Chambers, Henry F; Fowler, Vance G; Evans, Scott R; Patel, Robin; Klausner, Jeffrey D

    Background

    Pharyngeal and rectal Neisseria gonorrhoeae and Chlamydia trachomatis play important roles in infection and antibacterial resistance transmission, but no US Food and Drug Administration (FDA)-cleared assays for detection at these sites existed prior to this study. The objective was to estimate performance of assays to detect those infections in pharyngeal and rectal specimens to support regulatory submission.

    Methods

    We performed a cross-sectional, single-visit study of adults seeking sexually transmitted infection testing at 9 clinics in 7 states. We collected pharyngeal and rectal swabs from participants. The primary outcome was positive and negative percent agreement for detection of N. gonorrhoeae and C. trachomatis for 3 investigational assays compared to a composite reference. Secondary outcomes included positivity as well as positive and negative predictive values and likelihood ratios. Subgroup analyses included outcomes by symptom status and sex.

    Results

    A total of 2598 participants (79% male) underwent testing. We observed N. gonorrhoeae positivity of 8.1% in the pharynx and 7.9% in the rectum and C. trachomatis positivity of 2.0% in the pharynx and 8.7% in the rectum. Positive percent agreement ranged from 84.8% to 96.5% for different anatomic site infection combinations, whereas negative percent agreement was 98.8% to 99.6%.

    Conclusions

    This study utilized a Master Protocol to generate diagnostic performance data for multiple assays from different manufacturers in a single study population, which ultimately supported first-in-class FDA clearance for extragenital assays. We observed very good positive percent agreement when compared to a composite reference method for the detection of both pharyngeal and rectal N. gonorrhoeae and C. trachomatis.

    Clinical trials registration

    NCT02870101.