Rapid thermocycling methods

a nucleic acid and thermocycling technology, applied in the field of nucleic acid amplification methods and devices, can solve the problems of affecting the quality of nucleic acids, and affecting the quality of pcr assays, etc., and achieves the effect of rapid amplification of nucleic acids

Pending Publication Date: 2021-04-29
BIOFIRE DIAGNOSTICS LLC
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach enables faster and more efficient nucleic acid amplification with improved robustness and specificity, reducing cycle times and energy consumption while maintaining high yield, even with low template concentrations.

Problems solved by technology

Traditional microbiology techniques for diagnosing pathogens can take days or weeks, often delaying a proper course of treatment.
A challenge to using PCR as a primary means of diagnosis is the variety of possible causative organisms or viruses and the low levels of organism or virus present in some pathological specimens.
It is often impractical to run large panels of PCR assays, one for each possible causative organism or viruses, most of which are expected to be negative.
The problem may be exacerbated when pathogen nucleic acid is at low concentration and requires a large volume of sample to gather adequate reaction templates.
In some cases, there is inadequate sample to assay for all possible etiological agents.
While multiplex PCR has proven to be valuable in some systems, shortcomings exist concerning robustness of high level multiplex reactions and difficulties for clear analysis of multiple products.
However, this equilibrium paradigm does not fit well with physical reality.
Instantaneous temperature changes do not occur; it takes time to change the sample temperature, and temperature may not be homogeneous throughout the sample, particularly where larger volumes are used.
When PCR was first popularized in the late 1980s, the process was slow.
In combination, higher annealing and lower denaturation temperatures decrease the temperature range required for successful amplification.
Two-step cycling can, however, compromise polymerase extension rates if the combined annealing / extension step is performed at temperatures lower than the 70 to 80° C. temperature optimum where the polymerase is most active, particularly with fast ramp rates.
Even with protocol variations, amplification efficiency and yield are often poor when cycle times are <20 seconds when compared to control reactions.
As cycle times decrease from 20 seconds towards 2 seconds, PCR yield decreases and finally disappears, reflecting a lack of robustness even with simple targets at high copy number.
While performing two-step thermocycling in a closed container, one can rapidly thermocycle the small individual second-stage reactions, but it is much more difficult to thermocycle the large volume of the first-stage reaction fast enough to take advantage of the chemistries of U.S. Pat. No. 9,932,634.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Rapid thermocycling methods
  • Rapid thermocycling methods
  • Rapid thermocycling methods

Examples

Experimental program
Comparison scheme
Effect test

example 1

ity PCR

[0167]In one example, it is known that standard commercial immunofluorescence assays for the common respiratory viruses can detect seven viruses: adenovirus, PIV1, PIV2, PIV3, RSV, Influenza A, and Influenza B. A more complete panel illustratively would include assays for other viruses including: coronavirus, human metapneumovirus, rhinovirus, and non-HRV enterovirus. For highly variable viruses such as Adenovirus or HRV, it is desirable to use multiple primers to target all of the branches of the virus' lineage (illustratively 4 outer and 4 inner primer sets respectively). For other viruses such as coronavirus, there are 4 distinct lineages (229E, NL63, OC43, HKU1) that do not vary from one season to another, but they have diverged sufficiently enough that separate primer sets are required. The FilmArray® Respiratory Panel (BioFire Diagnostics, LLC of Salt Lake City, Utah) includes Adenovirus, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Human Meta...

example 2

with Reduced Volume

[0168]Sample volume reduction along with cycle time reduction was performed in an instrument and pouch similar to those shown in FIGS. 1-4, with a single Peltier functioning as heaters 886 and 887, to drive thermocycling of both of blisters 548 and 564 simultaneously. In this example, the Peltier was thermocycled as fast as possible unless otherwise indicated, approximately 12° C. / sec in heating and 9° C. / sec in cooling. In this example, the instrument and pouch were operated to provide a 140 μl volume of a sample that contained a PCR mixture including synthetic templates and primers for amplifying the synthetic templates in blister 546. All reactions in Example 2 were performed either using this synthetic mixture or a test pouch 580 that included actual assays for organisms. When the test pouch is used, a mixture of organisms is inserted in the pouch. After 6 cycles of PCR cycling with an annealing / extension hold of 15 seconds and a denature hold of 4 seconds, th...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Instruments, methods, and kits are disclosed for performing fast thermocycling.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of and priority to U.S. Provisional Patent Application. No. 62 / 681,830 filed Jun. 7, 2018, the entirety of which is incorporated herein by reference.BACKGROUND1. Technical Field[0002]Embodiments of the present disclosure relate generally to methods and devices for amplifying nucleic acids.2. Background[0003]In the United States, Canada, and Western Europe infectious disease accounts for approximately 7% of human mortality, while in developing regions infectious disease accounts for over 40% of human mortality. Infectious diseases lead to a variety of clinical manifestations. Among common overt manifestations are fever, pneumonia, meningitis, diarrhea, and diarrhea containing blood. While the physical manifestations suggest some pathogens and eliminate others as the etiological agent, a variety of potential causative agents remain, and clear diagnosis often requires a variety of assays be performed. Trad...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q1/68C12Q2527/107C12Q2527/109C12Q2527/113C12Q2563/143C12Q2563/149C12Q2565/629
InventorJONES, CHARLESHEMMERT, ANDREWCRISP, ROBERTCAMPBELL, ELIZABETH DIANAKIRK, KARINA S.HATCH, ANDREW CARTER
OwnerBIOFIRE DIAGNOSTICS LLC