US 12,442,047 B2
Methods for detecting Bordetella
Jules Chen, Irvine, CA (US); and Michelle Tabb, Santa Ana, CA (US)
Assigned to Quest Diagnostics Investments LLC, Secaucus, NJ (US)
Appl. No. 16/086,714
Filed by Quest Diagnostics Investments LLC, Secaucus, NJ (US)
PCT Filed Mar. 23, 2017, PCT No. PCT/US2017/023735
§ 371(c)(1), (2) Date Sep. 20, 2018,
PCT Pub. No. WO2017/165599, PCT Pub. Date Sep. 28, 2017.
Claims priority of provisional application 62/312,883, filed on Mar. 24, 2016.
Prior Publication US 2019/0382826 A1, Dec. 19, 2019
Int. Cl. C12Q 1/689 (2018.01)
CPC C12Q 1/689 (2013.01) [C12Q 2600/106 (2013.01); C12Q 2600/158 (2013.01); C12Q 2600/16 (2013.01)] 5 Claims
 
1. A method for detecting the presence of at least one pathogenic Bordetella species in an unprocessed biological sample comprising:
(a) contacting the biological sample with:
i. a first primer pair that amplifies an IS481 target nucleic acid consisting of SEQ ID NO: 1 or a complement thereof wherein the first primer consists of a first forward primer consisting of 5′ CCATAAGCATGCCCGATT 3′ (SEQ ID NO: 11) and a first reverse primer consisting of 5′ CGCTTCAGGCACACAAACT 3′ (SEQ ID NO: 10);
ii. a second primer pair that amplifies an IS1001 target nucleic acid consisting of SEQ ID NO: 2 or a complement thereof wherein the second primer consists of a second forward primer consisting of 5′ CGGCTCGACGAATTGC 3′ (SEQ ID NO: 7) and a second reverse primer consisting of 5′ AGTTCGTCACGCAGGACAT 3′ (SEQ ID NO: 8); and
iii a third primer pair that amplifies a hIS1001 target nucleic acid consisting of SEQ ID NO: 3 or a complement thereof, wherein the third primer pair consists of a third forward primer consisting of 5′ GGCACGGATCGAGGTTTTT 3′ (SEQ ID NO: 4) and a third reverse primer consisting of 5′ TACGGCCGTGAAGTGATAGA 3′ (SEQ ID NO: 5);
to produce a reaction-sample mixture under conditions where amplification of the IS481, IS1001, and hIS1001 target nucleic acids occurs if present in the biological sample, wherein the biological sample is not processed prior to amplification, and wherein the biological sample is a nasopharyngeal (NP) aspirate or wash, or a nasopharyngeal swab;
(b) subjecting the reaction-sample mixture to real-time multiplex PCR conditions under which each of the target nucleic acids present in the biological sample is amplified to produce a fluorescent signal;
(c) detecting the fluorescent signal generated by each amplified target nucleic acid produced in step (b); and
(d) detecting the presence of at least one pathogenic Bordetella species in the biological sample by evaluating the fluorescent signal of each target nucleic acid, whereby
i. B. holmesii is detected in the biological sample when a fluorescent signal is detected for the hIS1001 target nucleic acid;
ii. B. parapertussis is detected in the biological sample when a fluorescent signal is detected for the IS1001 target nucleic acid; and
iii. B. pertussis is detected in the biological sample when a fluorescent signal is detected for the IS481 target nucleic acid and no fluorescent signal is detected for the hIS1001 target nucleic acid;
wherein the pathogenic Bordetella species is one or more of B. pertussis, B. parapertussis, and B. holmesii.