Patent Application
20210324486
SARS-CoV-2 is a newly emerged virus that is presently the cause of enormous morbidity and mortality. There is a need in the art for methods for the sensitive and specific detection of SARS-CoV-2. This disclosure addresses that need.
The ASCII text file named “205961-7059US1(00207) Sequence Listing” created on Apr. 20, 2021, comprising 145 Kbytes, is hereby incorporated by reference in its entirety.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, exemplified materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
As used herein, the term “PCR” or “PCR amplification” refers to the polymerase chain reaction used to amplify nucleic acids. The term includes all variants of the polymerase chain reaction, including but not limited to quantitative PCR.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
The invention depends in part on the unexpected discovery of certain sites within the genome of SARS-CoV-2 that are useful binding sites for primers used in the detection of the virus. Accordingly, in one aspect the invention a method of detecting SARS-CoV-2 in a sample obtained from a subject, the method by contacting the sample with a composition comprising a plurality of PCR primers targeting at least one sequence selected from the group consisting of SEQ ID NOS: 1-559, thereby forming a PCR mixture; and conducting PCR amplification of the PCR mixture and detection of the at least one sequence selected from the group consisting of SEQ ID NOS: 1-559; wherein an elevated level of the at least one sequence selected from the group consisting of SEQ ID NOS: 1-559 indicates the presence of SARS-CoV-2 in the sample. In various embodiments, the sample is a blood sample or a saliva sample. In various embodiments, the method is a polymerase chain reaction (PCR) based method. In various embodiments the method further comprises analyzing the biological sample by quantitative PCR.
Below is a map of the SARS-CoV2-Wuhan-Hu-1 genome. The regions in capital boldface are the regions against which one can design good quality (sensitive and specific) q-PCR primers.
CATGTGGGCGAAATACCAGTGGCTTACCGCAAGGTTCTTCTTCGTAAGAACGGT
aataaaggagctggtggcca
CGCTATGTCGATAA
caacttctgtggccctgaTGGCTACCCTCTTGAGTGcattaaagaccttctagcacgtgc
CGTCACCAAATG
aatgcaaccaaatgtgcctttcaactctcatgaagtgtgatcattgtggtgaaacttcatgg
AGCATAGTCTTGCCGAATACCATAA
tgaatctggcttgaaaaccATTCTTCGTAAGGGTGGTCgcactattgcc
CTTG
attggcttgaagagaagtttaaggaaggtgtagagttTCTTAGAGACGGTTGGGAAAttgttaaatttat
ATGT
taacaaaggtgaagacattcaacttcttaagagtgcttatgaaaattttaatcagcacgaagttctactt
A
atgaaactcttgttacaatgccacttggctatgtaacacatggcttaaatttggaagaagctgctcggtatat
TAAGG
gtgtagaagctgttatgtacatgggcacactttcttatgaacaatttaagaaaggtgttcaGATACCTT
GTACGTGTGGT
aaacaagctacaaaatatctagtacaacaggagtcaccttttgttatgatgtcagcaccacct
AATGCGCAGGTAGCAAA
aagtcacaacattgctttgatatggaacgttaaagatttcatgtcattgtctgaaca
GATATTACGCACAACTA
atggtgactttttgcatttcttacctagagtttttagtgcagttggtaacatctgtt
AAGTTTACGCCCTGACACACGTTATG
tgctcatggatggctctattattcaatttcctaacacctaccttgaag
TGCGTAGTGA
tgtgCTATTACCTCTTACGCAATATAATAGatacttagctctttataataagtacaagtatttt
TTTGTTCGCATTCAAC
caggacagactttttcagtgttagcttgttacaatggttcaccatctggtgtttacca
CG
ccaagctttgttaaaaacagtacaattctgtGATGCCATGCGAAATGCTGGTattgttggtgtactgacatt
ACTACTATCGTTATA
atctaccaacaatgtgtgatatcagacaactactatttgtagttgaagttgttgataag
TTT
ctatagattagctaatgagtgtgctcaagtattgagtgaaatggtcatgtgTGGCGGTTCACTATATGTTA
GTAAAC
atttctcaatgATGATACTCTCTGACGATGCtgttgtgtgtttcaATAGCACTTATGCATCTCAaggt
AAAGCT
actgaggagacatttaaactgtcttatGGTATTGCTACTGTACGTGAAGTGCTGTCTgacagagaatt
CTAAA
aacagtaaagtacaaataggagagtacacctttgaaaaaggtgactatggtgatgctGTTGTTTACCGA
GGTACAA
caacttacaaattaaatgttggtgattattttgtgctgacatcacatacagtaatgccattaagtgc
TGCCGCTGTTGATG
cactatgtgagaaggcattaaaatatttgcctatagataaatgtagtagaattatacctg
TCTATGATGGGTTTTAA
aatgaattatcaagttaatggttaccctaacatgtttatcacccgcgaagaagctat
ACAGAGTCGTATTTG
tcttatgggcacatggctttgagttgacatctatgaagtattttgtgaaaataGGACCT
GAGCGCACCTGTTG
tctatgtgatagacgtgccacatgcttttccactgcttcagacacttatgcctgttggca
CAC
gttgcaatttaggtggtgctgtctgtagacatcatgctaatgagtacagattgtatctcgatgcttataac
AGTTAGGTGGT
ttacatctactgATTGGACTAGCTAAACGTTttaaggaatcaccttttgaattagaagatttt
ATAGA
tggttatgtcatgcatgcaaattacatattttggaggaatacaaatccaattcagttgtcttcctattc
GCGTGATCTCCC
tcagggtttttcggctttagaaccattggtagatttgccaataggtattaacatcactaggt
TTATTGTTGGCGTT
gcacttcttgctgtttttcagagcgcttccaaaatcataaccctcaaaaagagatggcaa
CGACGA
ctactagcgtgcctttgtaagcACAAGCTGATGAGTACGAACTTATGtactcattcgtttcggaaGAG
ACAGGTACGTTAATAGTtAATAGCGTACTTCTTTTT
cttgctttcgtggtattcttgctagttacactagccat
TTTACTCTCGTGT
taaaaatctgaattcttctagagttcctgatcttctggtctaaacgaactaaatattatat
TCGTAATCGGAGCTGTGATCC
ttcgtggacatcttcgtattgctggacaccatctaggacgctgtgacatcaag
ATTGATTAAACGAA
catgaaaattattcttttcttggcactGATAACACTCGCTACTTGTGAGCTTtatcacta
CA
tttcatcctctagctgataacaaatttgcactgacttgctttagcactcaatttgcttttgcttgTCCTGAC
GGCGTAAAACACGTCTATCAGTTACGTGCCAG
atcagtttcacctaaactgttcatcagacaagaggaagttca
ATCGGTAATTATACAGTT
tcctgtttaccttttacaattaattgccaggaacctaaattgggtagtcttgtagt
TGGCACCCGCAAT
cctgctaacaatgctgcaatcgtgctacaacttcctcaaggaacaacattgccaaaaggct
GTACGATCGAGTGTACAGTGAAC
aatgctagggagagctgcctatatggaagagccctaatgtgtaaaattaat
In various embodiments, the primers are selected from SEQ ID NOS: 1-559.
What follows describes a method for identifying primers that are:
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.
While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No. 63/012,750 filed Apr. 20, 2020. The entire content of this application is hereby incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63012750 | Apr 2020 | US |
