Development of Conventional and Real-Time Nucleic Acid Sequence-Based Amplification Assays for Detection of Chlamydophila pneumoniae in Respiratory Specimens

Abstract

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Chlamydophila pneumoniae 16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon. C. pneumoniae RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type C. pneumoniae RNA and 0.1 inclusion-forming unit (IFU) of C. pneumoniae. In spiked respiratory specimens, the sensitivity of the C. pneumoniae NASBA assay varied between 0.1 and 1 IFU/100 l sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.Chlamydophila pneumoniae is a common etiologic agent of respiratory tract infections in humans and is responsible for 3% of all cases of community-acquired pneumonia and 43% of all cases during an isolated miniepidemic (8,13,16,23,24). It has been associated with asthma (12), chronic obstructive pulmonary disease (21), chronic pharyngitis (9), a wide range of mild to serious extrapulmonary complications (11), and atherosclerosis (5).In the past, diagnosis of infection by this organism was based on the detection of fourfold rises in antibody titers in the microimmunofluorescence test or an elevated immunoglobulin G (IgG) or IgM titer when only a single serum sample was available. Culture confirmation of the diagnosis of C. pneumoniae infection, followed by the propagation of clinical isolates, has proven difficult to date. Therefore, nucleic acid amplification techniques have been introduced. PCR was shown to be considerably more sensitive than culture for the detection of C. pneumoniae (1, 2).Real-time nucleic acid sequence-based amplification (NASBA; bioMérieux, Boxtel, The Netherlands) is targeted at RNA. NASBA makes use of the simultaneous enzymatic activities of avian myeloblastosis virus reverse transcriptase, RNase H, and T7 RNA polymerase and molecular beacons (15) under isothermal conditions.The whole process of amplification and detection runs in a fluorescent reader. The same amplicons can also be used for conventional electrochemiluminescence (ECL) detection (14). The real-time NASBA technique has already been successfully applied for the quantification of human immunodeficiency virus type 1 isolates (7) and for the detection of Mycoplasma pneumoniae (18).The aim of this study was to develop both a conventional assay and a real-time NASBA assay for the detection of C. pneumoniae in respiratory specimens based on NASBA amplification of a 16S rRNA target sequence by using the NucliSens basic kit (10, 17) and to compare the results of the two assays. MATERIALS AND METHODSBacterial strains. C. pneumoniae (ATCC VR-1355) was grown on HEp-2 cells. Shell v...

Development of Real-Time Multiplex Nucleic Acid Sequence-Based Amplification for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae , and Legionella spp. in Respiratory Specimens

“…Fifteen M. pneumoniae PCR-positive specimens were negative by both real-time mono and multiplex NASBAs. However, seven of these specimens were U1A negative and thus contained no RNA (samples 19,20,23,24,29,32, and 33 [ Table 3]). For three patients (patients 23, 25, and 26 [ Table 3]), the PCR result was confirmed by a positive IgM result; one of these patients (patient 23 [ Table 3]) had a second specimen positive for M. pneumoniae, by both real-time mono and multiplex NASBAs.…”

“…in respiratory specimens based on 16S rRNA. The P1 primers and the beacons used were described previously (18,19,20). To reach a satisfactory sensitivity for all three pathogens, a generic P2 primer was designed to allow the simultaneous amplification of M. pneumoniae, C. pneumoniae, and as many Legionella spp.…”

“…For real-time multiplex NASBA, besides the species-specific P1 primers described previously (18,19,20), a generic P2 primer, 5Ј-GATGCAAGGTCGCA TATGAGAATTTGATCCTGGCTCAG-3Ј, was designed. The three P1 primers and this generic P2 primer were used in the real-time multiplex NASBA reaction.…”

“…Real-time mono NASBA reactions were performed using the NucliSens Basic Kit amplification module (bioMérieux) as described previously (18,19,20). In negative-control reactions, target nucleic acid was replaced by RNase-/DNase-free water.…”

“…The plasmids were transfected in Escherichia coli DH5␣ and used for large-scale generation of runoff transcripts after linearization with BamHI (Pharmacia Biotech). In vitro RNA was generated from these constructs with T7 RNA polymerase (Pharmacia Biotech) as described previously (16,19,20).…”