Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay.
Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5'-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease.
Pneumonia Response and Surveillance Laboratory, Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA, 30329, USA.
Kristen E
KE
Cross
Pneumonia Response and Surveillance Laboratory, Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA, 30329, USA.
Jeffrey W
JW
Mercante
Pneumonia Response and Surveillance Laboratory, Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA, 30329, USA.
Alvaro J
AJ
Benitez
Pneumonia Response and Surveillance Laboratory, Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA, 30329, USA.
Ellen W
EW
Brown
Pneumonia Response and Surveillance Laboratory, Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA, 30329, USA.
Maureen H
MH
Diaz
Pneumonia Response and Surveillance Laboratory, Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Atlanta, GA, 30329, USA. Electronic address: zdx2@cdc.gov.
Jonas M
JM
Winchell
D017931:DNA Primers / Q000235:genetics*; D006801:Humans; D007875:Legionella / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007877:Legionnaires' Disease / Q000382:microbiology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015345:Oligonucleotide Probes
Legionella; Legionella anisa; Legionella bozemanii; Legionella longbeachae; Legionella micdadei; Multiplex real-time PCR