Assay devices and methods of use thereof
A multifunctional chamber, stage technology, applied in the fields of biochemical equipment and methods, chemical instruments and methods, heating or cooling equipment, etc., which can solve the problem of high level multiple reaction robustness and difficulty in clearly analyzing multiple products.
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Embodiment 1
[0480] Example 1: High Density PCR
[0481]In one embodiment, standard commercial immunofluorescence assays for common respiratory viruses are known to detect seven viruses: adenovirus, PIV1, PIV2, PIV3, RSV, influenza A, and influenza B. A more complete panel illustratively includes assays for other viruses, including coronaviruses, human metapneumoviruses, rhinoviruses, and non-HRV enteroviruses. For highly variable viruses, such as adenovirus or HRV, multiple primers are required to target all branches of the viral lineage (eg, 4 outer and 4 inner primer sets, respectively). For other viruses, such as coronaviruses, there are 4 distinct lineages (229E, NL63, OC43, HKU1) that do not change from season to season, but are sufficiently differentiated to require separate primer sets. The Respiratory Panel (BioFire Diagnostics, LLC of Salt Lake City, UT) includes adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, human metapneumovirus, human rhi...
Embodiment 2
[0482] Example 2: Rapid PCR
[0483] use Figure 25-Figure 27D A prototype instrument constructed of bag and heater was used to amplify DNA. A 75 μl sample contains 10,000 copies of a synthetic DNA molecule of 110 bp and a 10-fold concentration of primers (5 μM each) and DNA polymerase (2 U / μL) (compared to standard PCR concentrations, as described in US 2015-0118715, which have been Incorporated by reference), and 0.45 mM dNTPs and 5 mM Mg++. 1X LCGreen was used for detection. It should be understood that the reaction mixture is exemplary only. Depending on the cycle time, elevated primer and polymerase concentrations may be beneficial. See US Patent Application No. 2015-0118715, which has been incorporated by reference, for more information on increased primer and polymerase concentrations. For example, for cycle times less than 20 seconds, it is desirable to have at least 0.5 μM of polymerase and at least 1 μM of each primer (in multiplex reactions) or at least 2 μM of...
Embodiment 3
[0486] Example 3: Three-temperature PCR using two temperature zones
[0487] As noted above, some PCR protocols use three temperatures: a first temperature for annealing, a slightly higher temperature for extension exemplarily selected based on enzymatic activity, and a third highest temperature for denaturation. While one heater is typically thermally cycled for three temperatures, in some embodiments it may be desirable to thermally cycle larger volumes quickly. Illustratively, first stage PCR may need to be thermally cycled through three temperatures, where a heater such as heater assembly 988 may not be able to heat and cool the contents of bubble 564 as quickly as desired.
[0488] In one such embodiment, it may be desirable to figure 1 The bag 510 uses a three-step PCR protocol in the first-stage PCR. As mentioned above, the figure 2 The first stage heater 886 is positioned to heat and cool the contents of the bubble 564 for the first stage PCR. In one embodiment, ...
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Abstract
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