WO2000036146A1

WO2000036146A1 - Method for the selective identification of salmonella

Info

Publication number: WO2000036146A1
Application number: PCT/GB1999/004271
Authority: WO
Inventors: Benjamin Wicks; Neil Percy; Richard Harley Grenville Owen
Original assignee: Celsis International Plc
Priority date: 1998-12-15
Filing date: 1999-12-15
Publication date: 2000-06-22
Also published as: AU1870200A; GB9827585D0

Abstract

A method for the detection of Salmonella in a sample, comprises contacting the sample with a polynucleotide capable of hybridising within the sequence shown in SEQ ID NO.1, and determining hybridisation.

Description

METHOD FOR SELECTIVE IDENTIFICATION OF SALMONELA

Field of the Invention

This invention relates to method for the selective identification of microorganisms, especially Salmonella. Background of the Invention

There are many commercial products which require testing for microbiological quality before they can be released for sale. Examples include food products, beverages, pharmaceutical preparations, and personal care products such as toothpaste, cosmetics and shampoos. Typically, microbiological quality standards include limits on the total number of microorganisms which may be present in a sample and/or limits on the numbers or presence/absence of specific types of organisms.

Testing is usually carried out on raw materials taken at various stages in the manufacturing process and on the finished products. Detection involves a series of culture steps including pre-enrichment, selective enrichment, selective plating and confirmation tests to identify the presence of the relevant microorganism.

Nucleic acid probes may be used in place of or in addition to these culture techniques, in order to facilitate the identification of specific microorganisms. For example, nucleic acid probes for the detection of specific microorganisms or groups of microorganisms have been designed based on the gene for 16S rRNA. The product of this gene is ubiquitous, being present in all organisms. However, one of the difficulties is that these probes are not always specific and can cross-react, or cross-hybridise, to the genes in other microorganisms. For example, probes based on 16S rRNA, designed for the detection of Salmonellae, have been shown to cross- react with non-Salmonella species which are not considered hazardous to health. This gives rise to false indications of contamination which makes it necessary to re-test samples which have indicated positive for Salmonella. In addition, the probes do not react with all Salmonellae and therefore, in certain circumstances, contamination with certain species will not be detected. US-A-5792854 discloses the detection of Salmonella, using probes that hybridise to defined regions of the gene for 16S or 23S rRNA. It is acknowledged in US-A-5792854 that a universal Salmonella probe may be unattainable. The 23S rRNA region 512-560 is acknowledged as representing a particular problem; regions 1405-1597 and 1700-1760 are preferred, in addition to certain regions of 16S rRNA. Summary of the Invention

The present invention is based on the discovery of polynucleotide molecules capable of hybridising selectively to the 23S rRNA gene of Salmonella, and which do not hybridise to the same gene in a microorganism of a different genus. Such a polynucleotide molecule has a nucleic acid sequence that hybridises within the sequence shown as SEQ ID NO. 1.

Without wishing to be bound by theory, the 23S rRNA gene may provide more sequence information than the 16S rRNA gene and so may provide additional regions that are specific for one type of microorganism but which lack homology in other microorganisms. Now, a surprising degree of specificity has been found within 23S rRNA. Description of the Invention

SEQ ID NOS. 1 and 2 represent two regions of the 23S rRNA gene sequence from Salmonella typhimurium which appear to be highly conserved in different Salmonella species but which lack significant homology in other microorganisms. Part at least of SEQ ID NO. 2 is essentially the same as disclosed as regions 1405-1597 and 1700-1760 in US-A-5792854; by comparison, SEQ ID NO. 1 provides a useful source for the design of probes having high specificity for Salmonella but low specificity for other microorganisms, under the hybridisation conditions used. Probes having this specificity are shown in SEQ ID NOS. 3 and 4.

Hybridisation assays and probes are known to those skilled in the art, and are described in US-A-5792854, the relevant content of which is incorporated herein by reference. Suitable probes for use in this invention are typically of between 15 and 25 nucleic acids in length, e.g. about 20 nucleic acids. They are preferably chosen so that they are identical or complementary to a part of SEQ ID NO. 1 , in either sense. However, any probe that exhibits hybridisation under stringent conditions, without exhibiting self-complementarity, may be used. By way of example only, the probes of SEQ ID NOS. 3 and 4 are exactly complementary to nucleic acids 292-313 and 322-343 of SEQ ID NO. 1. The following Example illustrates the invention.

Example

A test panel of Salmonella microorganisms included strains of pathogenic Salmonella. A non-target group was chosen to include species closely related to Salmonella, including those that are difficult to distinguish from Salmonella on the basis of growth characteristics alone and require biochemical or serological profiling.

Chromosomal DNA was isolated from the individual strains. DNA oligonucleotide primers were designed to enable the 23S ribosomal RNA genes of the chosen organisms to be amplified by the polymerase chain reaction, as described in Van Camp etal, Current Microbiology 27:147-151 (1993). The amplified rDNA genes were purified to remove contaminating PCR primers, and the nucleotide sequences determined.

Having obtained the DNA sequences from the different species, the sequences were aligned to identify those regions that were homologous to different species within the genus Salmonella. Analogous regions in the non-target organisms were then examined to determine the presence of differences which would be useful in the design of nucleic acid probes for binding to Salmonella species only.

Two regions, shown in SEQ ID NO. 1 and 2, were initially identified as being conserved within the Salmonella genus but containing significant variations in all of the other closely related species tested. These regions can be used to discriminate between Salmonella and non-Salmonella species using known nucleic acid technologies such as probe hybridisation and PCR.

Based on these respective regions, the oligonucleotide probes shown as SEQ ID NOS. 3, 4, 5 and 6 were designed. They and 16S probes were used in a test hybridisation assay, to evaluate their specificity.

Probes were used in pairs, those of SEQ ID NOS. 3 and 5 being the capture probes and those of SEQ ID NOS. 4 and 6 being labelled, as detector probes.

Results are shown in the Tables. "Sal cap/det" refers to 16S rRNA probes; "23S 300/300" and "23S 1700/1700" respectively refer to the probes of the invention and those of SEQ ID NOS. 5 and 6.

Table 1 shows that the 16S probes detected all 5 Salmonellae but also detected 3 non-target organisms; the 23S 300 set detected all Salmonellae and no non-targets; and the 23S 1700 set detected all Salmonellae and had a high signal for K. planticola. Thus, while both 23S sets detected all the Salmonella serotypes, the

300 pair shows better specificity than the 1700 pair.

Table 2A shows that the 1700 pair detects all Salmonellae, but detection of Salmonella arizona is extremely poor. Table 2B shows that the 300 pair detects all Salmonellae including S. arizona, and is clearly better than the 1700 pair.

Table 3 shows that the 300 pair weakly detects 2 false positives strains. The 1700 pair strongly detected 6 non-Salmonella strains.

Table 1

Signal minus BG

Ref

No Strain Probes 10μl_ 1μL BG

1 Salmonella typhimurium Sal cap / det 23434 8368 151

64 Salmonella pullorum Sal cap / det 2886 1402 151

74 Salmonella gallinarum Sal cap / det 212 359 151 75 Salmonella a bony Sal cap / det 17243 11158 151 79 Klebsiella planticola Sal cap / det -29 -88 151 81 Enterobacter dissolvens Sal cap / det 19494 5288 151

144 Escherichia vulneris Sal cap / det -4 -30 151

43 Escherichia coli Sal cap / det -28 15 151

32 Enterobacter cloacae Sal cap / det 16084 5650 151

148 Escherichia adecarboxylata Sal cap / det 37667 8321 151

30 Citrobacter freundii Sal cap / det 81 -3 151

1 Salmonella typhimurium 23 S (300/300) 229631 63142 696

64 Salmonella pullorum 23 S (300/300) 36461 23308 696

74 Salmonella gallinarum 23 S (300/300) 302 941 696 75 Salmonella abony 23 S (300/300) 241479 235863 696 79 Klebsiella planticola 23 S (300/300) 216 -135 696 81 Enterobacter dissolvens 23 S (300/300) -146 -181 696

144 Escherichia vulneris 23 S (300/300) 0 67 696

43 Escherichia coli 23 S (300/300) 122 220 696

32 Enterobacter cloacae 23 S (300/300) 207 -122 696

148 Escherichia adecarboxylata 23 S (300/300) -188 -99 696

30 Citrobacter freundii 23 S (300/300) -22 -68 696

1 Salmonella typhimurium 23 S (1700/1700) 431071 185365 537

64 Salmonella pullorum 23 S (1700/1700) 36540 4380 537

74 Salmonella gallinarum 23 S (1700/1700) 240872 1026 537 75 Salmonella abony 23 S (1700/1700) 273276 72387 537 79 Klebsiella planticola 23 S (1700/1700) 275 948 537 81 Enterobacter dissolvens 23 S (1700/1700) -66 10 537

144 Escherichia vulneris 23 S (1700/1700) 61 117 537

43 Escherichia coli 23 S (1700/1700) 157 208 537

32 Enterobacter cloacae 23 S (1700/1700) 101 433 537

148 Escherichia adecarboxylata 23 S (1700/1700) -20 20 537

30 Citrobacter freundii 23 S (1700/1700) -20 0 537 Table 2A

Table 2B

Table 3

Signal minus

BG

Claims

1. A method for the detection of Salmonella in a sample, which comprises contacting the sample with a polynucleotide capable of hybridising within the sequence shown in SEQ ID NO. 1, and determining hybridisation.

2. A method according to any preceding claim, wherein the polynucleotide comprises the sequence shown in SEQ ID NO. 3 or 4.

3. A method according to claim 1 or claim 2, wherein the sample is a food product, a personal care product, a pharmaceutical product or water.