A method for detecting the presence of at least one pathogenic Bordetella species in a biological sample comprising: (a) contacting the biological sample with: i. a first primer pair that amplifies an IS481 target nucleic acid comprising nucleotides that are at least 85-95% identical to SEQ ID NO: 1 or a complement thereof; ii. a second primer pair that amplifies an IS 1001 target nucleic acid comprising nucleotides that are at least 85-95% identical to SEQ ID NO: 2 or a complement thereof; and iii. a third primer pair that amplifies a hIS 1001 target nucleic acid comprising nucleotides that are at least 85-95% identical to SEQ ID NO: 3 or a complement thereof; to produce a reaction-sample mixture under conditions where amplification of the IS481, IS1001, and hIS1001 target nucleic acids, if present in the biological sample, occurs without extracting the target nucleic acids from the biological sample; (b) subjecting the reaction-sample mixture to real-time 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; wherein the biological sample is not subjected to an extraction or purification step prior to amplification; and wherein the biological sample is a nasopharyngeal aspirate, wash, or nasal swab. (a) contacting the biological sample with: i. a first primer pair that amplifies an IS481 target nucleic acid comprising nucleotides that are at least 85-95% identical to SEQ ID NO: 1 or a complement thereof; ii. a second primer pair that amplifies an IS 1001 target nucleic acid comprising nucleotides that are at least 85-95% identical to SEQ ID NO: 2 or a complement thereof; and iii. a third primer pair that amplifies a hIS 1001 target nucleic acid comprising nucleotides that are at least 85-95% identical to SEQ ID NO: 3 or a complement thereof; to produce a reaction-sample mixture under conditions where amplification of the IS481, IS1001, and hIS1001 target nucleic acids, if present in the biological sample, occurs without extracting the target nucleic acids from the biological sample; i. a first primer pair that amplifies an IS481 target nucleic acid comprising nucleotides that are at least 85-95% identical to SEQ ID NO: 1 or a complement thereof; ii. a second primer pair that amplifies an IS 1001 target nucleic acid comprising nucleotides that are at least 85-95% identical to SEQ ID NO: 2 or a complement thereof; and iii. a third primer pair that amplifies a hIS 1001 target nucleic acid comprising nucleotides that are at least 85-95% identical to SEQ ID NO: 3 or a complement thereof; (b) subjecting the reaction-sample mixture to real-time 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; wherein the biological sample is not subjected to an extraction or purification step prior to amplification; and wherein the biological sample is a nasopharyngeal aspirate, wash, or nasal swab. 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;