Molecular Detection and Quantification of Enterococci

Abstract

A primer pair and probe for the large subunit ribosomal RNA gene of enterococci for use in a real-time nucleic acid sequence based amplification (NASBA) assay.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

The FIGURE shows sequences for primers and probe for NASBA amplification of Enterococci.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

NASBA primers and probes were designed based on homologous regions of the large subunit ribosomal RNA gene (The FIGURE). This region is highly conserved within all reported species of the Enterococcus genus (Haugland et al., 2005). Previous researchers (Frahm and Obst, 2003) used this region as the priming site for the development of a Taqman PCR assay for enterococci detection. The primers have been modified to best fit the NASBA amplification requirements and molecular beacon design criteria as well as minimize the cross hybridization to each other. This primer set has been tested against Enterococcus faecalis and E. faecium, and E. avium, as well as several other genera of Gram positive and Gram negative bacteria to determine the specificity of the assay and ensure that no cross reactivity occurs to non-target organisms. Data thus far indicate that the primer set is specific to the genus enterococci.

The assay sensitivity has been evaluated using serially diluted RNA isolated from enterococci cells. It is also possible to include cell extract samples from individual species as well as mixtures of all of the enterococci species available. Because ribosomal RNA tends to have a complex secondary structure, the NASBA assay was initiated by a 65° C. denaturing step for 3 minutes prior to the 41° C. amplification step (60 to 90 minutes). This initial denaturation is required for some, but not all NASBA assays and its requirement for efficient amplification of enterococci still needs to be evaluated.

Amplification inhibitors are often co-purified with natural environmental samples. We have encountered such inhibitors in our coastal water filtrates (Casper et al., 2005b). However, this inhibition tends to occur in sample volumes greater than 200 ml. For enterococci samples, the sample volume will always be less than 100 ml (as used by the DOH). Also, we will include an internal control (IC-NASBA protocol) that will be used to normalize the amplification results (Patterson et al., 2005). This approach will increase the precision of our quantification and reduce the amount of false negative results. Because the internal control molecule contains the same priming site as the target, the amplification reaction becomes competitive. Serial titrations of internal control RNA determine the optimal concentration to allow for consistent quantification of enterococci within the expected environmental concentrations (Patterson et al, 2005).

REFERENCES

The following citations are incorporated herein by reference:

• Haugland R A, Siefring S C, Wymer L J, Brenner K P, Dufour A P. 2005. Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis. Water Res. 39(4):559-68.

Frahm E, Obst U. 2003. Application of the fluorogenic probe technique (TaqMan PCR) to the detection of Enterococcus spp. And Escherichia coli in water samples. J Microb Meth 52: 123-131.

It will be seen that the advantages set forth above, and those made apparent from the foregoing description, are efficiently attained and since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween. Now that the invention has been described,

Claims

1. A method for quantifying Enterococci in a sample of saline water, said method comprising: performing NASBA amplification, wherein said amplifying step comprises contacting said sample with a pair of primers to produce an amplification product, wherein said pair of primers comprises a first primer and a second primer, wherein said first primer comprises SEQ ID NO:1, and said second primer comprises SEQ ID NO: 2.

2. The method of claim 1 wherein said second primer includes a T7 promoter region comprising SEQ ID NO: 3.

3. The method of claim 1 further comprising a probe, said probe comprising SEQ ID NO: 4.