Detection of Legionella

Details Detection of Legionella Detection of Legionella

2002-05-07

2004-12-14

Riley, Jezia (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C435S183000, C536S024300, C536S024320, C536S024330

Reexamination Certificate

active

06830888

ABSTRACT:

TECHNICAL FIELD
This invention relates to bacterial diagnostics, and more particularly to detection of
Legionella
species, particularly
Legionella pneumophila.
BACKGROUND
The genus
Legionella
, family
Legionellaceae
, includes over 40 different species of fastidious gram-negative bacilli, with over 60 described serogroups. While some of these organisms represent normal environmental flora, many have been shown to cause human disease, namely opportunistic pneumonia in immunocompromised patients. The vast majority of such cases (approximately 85%) are due to
L. pneumophila
, with the remainder due to other species, most commonly
L. micdadei, L. bozemanii, L. dumoffii
, and
L. longbeachae. Legionella pneumonia
can be community acquired or nosocomial, and sporadic or epidemic in nature. Pulmonary infection may be subclinical, or severe and life threatening. The fatality rate can approach 50% in immunocompromised patients. The organism often responds to antimicrobial therapy, usually with macrolides, and clinical responses usually occur within 3-5 days. The latter fact, combined with clinical and radiographic features that are often non-specific, serve to underscore the value of a prompt and accurate laboratory diagnosis.
SUMMARY
The invention provides for methods of identifying
Legionella
in a biological sample, and further, for specifically detecting
Legionella pneumophila
. Primers and probes for detecting
Legionella
, specifically
L. pneumophila
, are provided by the invention, as are kits containing such primers and probes. Methods of the invention can be used to rapidly identify
Legionella
nucleic acids from specimens for diagnosis of
Legionella
infection. Using specific primers and probes, the methods include amplifying and monitoring the development of specific amplification products using real-time PCR.
In one aspect of the invention, there is provided a method for detecting the presence or absence of
Legionella
in a biological sample from an individual. The method to detect
Legionella
includes performing at least one cycling step, which includes an amplifying step and a hybridizing step. The amplifying step includes contacting the sample with a pair of 5S rRNA primers to produce a 5S rRNA amplification product if
Legionella
nucleic acid encoding 5S rRNA is present in the sample, and the hybridizing step includes contacting the sample with a pair of 5S rRNA probes. Generally, the members of the pair of 5S rRNA probes hybridizes to the amplification product within no more than five nucleotides of each other. A first 5S rRNA probe of the pair of 5 S rRNA probes is typically labeled with a donor fluorescent moiety and a second 5S rRNA probe of the pair of 5S rRNA probes is typically labeled with a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of fluorescent resonance energy transfer (FRET) between the donor fluorescent moiety of the first 5S rRNA probe and the acceptor fluorescent moiety of the second 5S rRNA probe. The presence of FRET is usually indicative of the presence of
Legionella
in the biological sample, while the absence of FRET is usually indicative of the absence of
Legionella
in the biological sample. In addition, determining the melting temperature between one or both of the 5S rRNA probe(s) and the corresponding probe targets can confirm the presence or absence of the
Legionella
.
In another aspect, the invention features a method for detecting the presence or absence of
L. pneumophila
in a biological sample from an individual. The method to detect
L. pneumophila
includes performing at least one cycling step, which includes an amplifying step and a hybridizing step. The amplifying step includes contacting the sample with a pair of mip primers to produce a mip amplification product if
L. pneumophila
nucleic acid encoding mip is present in the sample, and the hybridizing step includes contacting the sample with a pair of mip probes. Generally, the members of the pair of mip probes hybridizes to the amplification product within no more than five nucleotides of each other. A first mip probe of the pair of mip probes is typically labeled with a donor fluorescent moiety and a second mip probe of the pair of mip probes is typically labeled with a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of FRET between the donor fluorescent moiety of the first mip probe and the acceptor fluorescent moiety of the second mip probe. The presence of FRET is usually indicative of the presence of
L. pneumophila
in the biological sample, while the absence of FRET is usually indicative of the absence of
L. pneumophila
in the biological sample. The method to detect
L. pneumophila
can be performed after the method has been performed to detect
Legionella
or concurrent with the method to detect
Legionella
.
A pair of 5S rRNA primers generally includes a first 5S rRNA primer and a second 5S rRNA primer. The first 5 S rRNA primer can include the sequence 5′-ACT ATA GCG ATT TGG AAC C-3′ (SEQ ID NO: 1), and the second 5S rRNA primer can include the sequence 5′-GGC GAT GAC CTA CTT TC-3′ (SEQ ID NO:2). The first 5S rRNA probe can include the sequence 5′-CAT GAG GAA GCC TCA CAC TAT CA-3′ (SEQ ID NO:3), and the second 5S rRNA probe can include the sequence 5′-GGC GAT GAC CTA CTT TC-3′ (SEQ ID NO:2). In certain aspects, the second 5S rRNA primer can be labeled with a donor fluorescent moiety and can act as the second 5S rRNA probe.
A pair of mip primers generally includes a first mip primer and a second mip primer. The first mip primer can include the sequence 5′-ACC GAA CAG CAA ATG AAA GA-3′ (SEQ ID NO:4), and the second mip primer can include the sequence 5′-AAC GCC TGG CTT GTT TTT GT-3′ (SEQ ID NO:5). The first mip probe can include the sequence 5′-AAC AAG TTT CAG AAA GAT TTG ATG GCA AAG-3′ (SEQ ID NO:6), and the second mip probe can include the sequence 5′-GTA CTG CTG AAT TCA ATA AGT AAG CGG ATG-3′ (SEQ ID NO:7).
The members of the pair of 5S rRNA probes can hybridize within no more than two nucleotides of each other, or can hybridize within no more than one nucleotide of each other. A representative donor fluorescent moiety is fluorescein, and representative acceptor fluorescent moieties include LC™-RED 640 (LightCycler™-Red 640-N-hydroxysuccinimide ester), LC™-RED 705 (LightCycler™-Red 705-Phosphoramidite), and cyanine dyes such as CY5 and CY5.5.
In one aspect, the detecting step includes exciting the biological sample at a wavelength absorbed by the donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by the acceptor fluorescent moiety. In another aspect, the detecting includes quantitating the FRET. In yet another aspect, the detecting step is performed after each cycling step, and further, can be performed in real-time.
Generally, the presence of the FRET in an amount at least 3 times the amount of FRET in a sample lacking the
Legionella
5S rRNA nucleic acid molecule indicates the presence of a
Legionella
infection in the individual. Representative biological sample include sputum, bronchio-alveolar lavage, bronchial aspirates, lung tissue, urine and blood.
The above-described methods can further include preventing amplification of a contaminant nucleic acid. Preventing amplification can include performing the amplifying step in the presence of uracil and treating the biological sample with uracil-DNA glycosylase prior to a first amplification step. In addition, the cycling step can be performed on a control sample. A control sample can include a portion of the
Legionella
nucleic acid molecule encoding 5S rRNA. Alternatively, such a control sample can be amplified using a pair of control primers and hybridized using a pair of control probes. The control primers and the control probes are usually other than the 5S rRNA primers and 5S rRNA probes, respectively. A control amplification product is produced

Affiliated with

Also associated with

Rating

Detection of Legionella does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Detection of Legionella, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Detection of Legionella will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3332181