AP98A - Development of a salmonella-specific probe for rapid routine identification and diagnosis of salmonella SPP in foods. - Google Patents

Abstract

The invention relates to a DNA fragment derived from Salmonella DNA and which may be used for the detection of Salmonellae spp. in for example a food or clinical sample. The invention also relates to a method of screening for the presence of Salmonellae spp. in such a sample using the DNA fragment of the invention.

Description

This invention relates to a DNA fragment for use in the detection of Salmonellae spp. in for example food or clinical samples. The invention also relates to a method of detecting the presence of Salmonellae spp. in such samples using the DNA fragment.

The presence of enterobacteriaceae (Salmonella , Shigella,

Yersinia, Pseudomonas, toxigenic Escherichia, etc.) in various types of foods is a worldwide health hazard. In the Third World, chronic infections with several species of enterobacteriaceae including Salmonellae spp., persist due to inadequate detection and diagnostic methods.

Conventional methods for isolating, and detecting Salmonellae spp. rely in part, on the use of highly selective enrichment conditions and plating media, to detect Salmonella, which are usually found in the the presence of high levels of competing bacteria. The method of preenrichment has to specify two selective enrichment broths and a minimum of two plating media must also be specified (F.D.A., 1976: Poeima and Silliker, 1976). These conditions impose a time-factor which is undesirable in cases of severe diarrhoea which needs immediate attention.

Conventional methods often generate false positive as well as false negative results.

According to one conventional method, samples are brought into the laboratory and incubated in selenite-lactose enrichment broth or selective tetrathionate broth for 24 hours at 37°C. After this initial incubation in selective media, the bacteria are plated onto indicator media (green-lactose-sucrose-phenol red agar), and further incubated for 24 hours at 37°C. Suspect colonies on the indicator media are subinnoculated by stabbing and streaking a butt/slant each of Kl.igler iron-lactosc-dextrose phenol red, lysine iron-dextrose-bromo cresol purple and urea-dextrose-phenol red agar. This is then incubated tor another l8 hours at 37°C- Again suspect cultures after this incubation

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-2are tested against polyvalent 0 and II (Welcome Ltd) antisera before they arc finally submitted to a laboratory for typing. This monitoring programme, using the conventional method is not only slow but gives unreliable results as it generates a lot of false results. In most cases of Salmonella infection in diarrhoea, enteric fevers and food poisoning, Salmonella is often found in the presence of competing enterobactereaceae which cause similar disease symptons. In such cases, it is not possible to detect presence of Salmonella in the presence of competitive bacteria using the classical microbiological method. A number of case studies have demonstrated that it is possible to detect the presence of one Salmonella cell only in cases where the sample being tested was pure and did not have competing bacteria (Edel and Kainpetmacher, 1969) · When large numbers of competing bacteria are present as much as two thirds of the bacteria present, must be Salmonella in order for the test to give positive results (Edel and Kampelmacher 1909)· Ibis demonstrates that, the conventional method is not only inefficient, but generates unreliable results since it generates high level of false negatives.

Often, acceptable government, industrial and international standards for the detection of Salmonellae spp. in foods, drinking water and animal feeds are not easy to meet especially with the facilities available in the Third World. For example, the American

Food and Drug Association standards for Salmonellae spp. in foods, and animal feeds specify that this organism must bo absent from as many as 6025g analy tical units drawn from a production lot (Olson 1975) ·

This level of sensitivity is not easy to attain with conventional methods of detecting· Salmonellae spp. since it· is often recommended that an overnight norwselective enrichment culture be followed by another overnight selective and indicator culture media (D¹ Aoust, andMaishment, 1979: A _____________BAD-0RK31NAL n®

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-3U.S.F.D.A. : 1978). A conventional analysis for Salmonellae spp.

following these methods would require a minimum of 5 days to complete, after sample receipt, to obtain presumptive results.

A number of researchers have developed screening procedures in an attempt to reduce the time required to complete a Salmonellae spp. * analysis. These methods include; fluorescent antibody staining a

(Silliker, Schmall, and Chiu, I960), enrichment serology (Danwart, and Kneitzer, 1969, Sperber and Deibel, 1969); enzyme-linked immunoassay (Krysenski and lleimsch, 1977); direct and indirect immunoassay which can be performed with or without the use of further subcultures (Thomason, 198l); impedance assays (Cady, Dufour, Show, and Knaeger,

1978) and radiometry (Stewart, Eyles and Murrell, 1980). Most of these methods are carried out either directly from the selective enrichment broth or from a post cnriclunent brot h which has been incubated f< r 24 hours. The majority of those methods also generate false positive and false negative results because of aberrant reactions due to crossreactivities. False positive results of 4% to 17% have been reported for the fluorescent antibody staining method (Insalata, Mahnke, and Dunlop 1973; Mohr, Trerk and Yelerian, 1974; Insalata, Dunlop and Mahnke, 1975; Munson, Slirade, Discullo, Fantasia, Hartung and O'Conno^, 1976: Thomason and Hebert, 1974 and Thomason, Hebert and Mahnke 1975)^

One other drawback is that the presumptive results obtained by these methods have to be confirmed using the conventional methods, which further confuse these screening procedures, especially when foods containing high levels of competing Gram-negative bacteria are to be evaluated.

Recent developments in recomb'nant DNA technology' have made possible the use of nOn-convention.h methods which employ cloned DNA sequences to detect, the presence of complementary DNA sequences among _____ BAD ORIGINAL dS

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- Page 4 a population of heterogeneous bacteria (Moseley, Hug, Alim,

So, Samodpour - Motalebi and Falkow, 1980; and Hill, 1981).

However, the identification and isolation of such a DNA sequence from any organism to be used as a probe, presents major problems.

Salmonellae spp. have no virulence factors or toxin genes equivalent to those found in E. coli or Y. enterocolitica, nor do they have a widely distributed plasmid or set of plasmid genes that can be used to detect the presence of all Salmonellae spp. It was therefore necessary to search the Salmonella genome for a chromosomal sequence that is not only specific to Salmonella but is present in all Salmonellae spp. could be surprising, giving the close biochemical relationship of Salmonellae spp. with other Enterobacteriaceae, which often produce similar disease symptoms. However, a number of such sequences have been demonstrated to exist in Salmonella by other workers (Fitts, Dismond, Hamilton and Neri, 1983).

According to one aspect of the invention there is provided a substantially pure DNA fragment from Salmonellae or alternatively

Salmonella-typhimurium comprising either:

a) The DNA sequence inserted into plasmid pNO 1523· The plasmid is carried by E. coli MC 1009 identified by accession no.

Z.G. SAL.15-DNA probe/88.

b) DNA sequences which will hybridize under low stringency conditions with the DNA sequence of Salmonellae.

The DNA fragment may be derived from S. typhimurium. Preferably, the DNA fragment is obtained by endonucleus enzyme restriction of the

S. typhimurium chromosomal DNA with lipa 1. The DNA fragment may be about 2.3· kb in length.

The DNA fragment may be Labelled in order to facilitate detection. The DNA fragment may be radiolabelled. Preferably the DNA fragment may be labelled with non-isotopic materials such as biotin or sulfoprobe-DNA detection system.

According to another aspect of the invention there is provided a pi asmid which contains an inserted DNA sequence in which the inserted sequence is a Ι5ΝΛ fragment of the invention. _____ _______________BAD ORIGINAL

- Page 5· According to another aspect of the invention there is provided a host cell transformed with a plasmid carrying a DNA fragment of the invention. The host cell may be a bacterial cell. Preferably the host cell is E. coli MC 1009·

According to a further aspect of the invention there is provided a method of screening for the presence of Salmonellac spp. and a sample, comprising adding a DNA fragment of the invention to the sample under hybridization conditions and establishing the presence or absence of SalmonelLae by hybridization of the DNA fragment with Salmonella DNA present in the sample.

The method may be carried out under low stringency hybridization conditions. Preferably, the method is carried out under high stringency hybridization conditions.

The invention also provides a Salmonella - specific DNA fragment which is essentially pure and capable of hybridizing to the DNA of all Salmonellae spp.

The production of a DNA probe in accordance with the invention and its use in the detection of Salmonellae spp. will now be described b way of example only with reference to the accompanying representations in which :

Fig 1. shows agarose gel electrophoretic separation of Sma I and lipa I restricted DNA from S. typhimurium compared with Eco RI and Hin d III restricted lambda DNA as a reference. This is a test of purity of DNA preparation used to make Salmonellae genomic library;

Fig 2 is a map of plasmid pNO 1523 showing the important Hpa I restriction site used in cloning;

Fig 3 illustrates the use of pNO 1523 as a cloning vector;

Fig 4 (a) shows an agarose gel electrophoretic separation of plasmid pNO 1523 DNA and recombinant pNO 1523 DNA containing inserts of ^xSr

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S. typhimurium DNA.

Fig 4(b) siiows restriction endonucleus pNO 1523 DNA with Hpa I.

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-()Fig 5 shows agarose gel electrophoretic separation ol' recombinant pNO 1523 DNA containing S . t, y p h i nmr i mn DNA inserts;

Fig 6 shows agarose gel electrophoretic separation of lipa I and

Sni a I linearized pNO J 5 2 3 and S 1, yph i inn r i 11111 DNA inserts;

Fig 7 is an autoradiograph of a hybridization experiment to select

SalmoncJ la - specific D N A fragments;

Fig 8 shows excised fragments of S. typh i inn r i mn DNA recovered from low melting temperature geLs after restriction with IIpa i;

Fig 9(a) shows the results colony hybridization between Salmonella

DNA probes and various bacterial species.

Fig 9(b), (c) and (d) show the results of colony hybridization

2 between P labelled probe from clone 15 and SaImonel Lae and other species of enterobacteriaceae;

Fig 10 shows the determination of the molecular weight of the

Hpa I digested S. typhimurium DNA fragment, and shows the molecular weight to be approximately 2,3Kb in length;

Fig 11 shows the results of dot blot hybridization between the DNA probe of the invention and mixed cultures of S. typhimurium and ShigeIla. This also demonstrates the sensitivity of the DNA probe.

Fig 12 shows the results of dot blot hybridizations between the DNA probe of the invention and mixed cultures of S, typhimurinm, E. coli, and Shigella, to further demonstrate the sensitivity of the probe and its ability to detect Sa I mono 1 lac in mixed population of bacteria;

Fig 13 shows the use of the Sa linone I La specific DNA probe of the invention in the detection of SalmoncI Iae spp. in meat and clinical samples.

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-7I. I1ETH0DS AND HATE RIALS

1.1. ISOLATION OF S. TYPHItiURIU!· AND E. COLI MC 600 HIGH KCLECULAr. WEIGHT CHR0I1CS0I1AL DNA

Salmonella typhimurium and E. coli MC 600 cells were harvested from liquid broth by centrifugation at 60G0 rpn for 20 r.iinutes and resuspended in a snail volume (20 ml) of 1C mli Tsis-Hcl Ihifl EDTA pH 8.0. When the cells v/ere completely suspended, more buffer ' was added to a total volume of 60 r.:l and 25% SDS was added to give a final concentration of 0,5% and the mixture’was vigorously shaken, then incubated at 37°C for 2 hours. Pronase (50 mc/i.;l) was added to each preparation toa final concentration of 50jug/ml and the solutions were incubated at 37°C for 15 hours. If the cells were not well lysed after the incubation period, more SDS was added to each preparation to bring toa final concentration of 1% followed by 15 minutes incubation at 60 C. This was followed by the addition of 0,5 ml isoar.ylalcohol to each lysate which were then mixed well by gentle shaking. Equal volumes of phenol - satuarated Tris-ETA (TE) were added to each preparation. The mixtures were then further shaken well for 30 minutes. 7he resultant emulsions were broken by centrifugation at 6000 rpm for 20 minutes^- The supernatants were recovered and their volumes measured. Sodium. & perchlorate (511) was added to each solution to afinal concentration of 1 It and the solutions were mixed well. Equal volumes of chloroform were added to each of the solutions and the solutions were further shaken for 15 minutes, then centrifuged at 6000 rpm for 20 minutes. The supernatants were recovered and their volumes were determined. The phenol chloroform steps v/ere repeated and 1 fl NaCl was added (0.1 II final concentration) to each of the final supernatants and the DMAs were precipitated by addition of 2 volumes (v/v) of cold ethanol (-20°C).

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-8The precipitated DMAs were spooled on glass rods and were each dissolved in 30 ml of Tris-EDTA buffer, pH 7,5.

1.2 RNASE . TREATMENT

Pancreatic RNase (10 i.ig/ml) was added (50 jjg/nl final concentration) to each DMA solution and incubated for one hour at 60°C. Pronase (50jug/ml final concentration), and SDS (0.5% final concentration) were added to t'ne solutions. The solutions were nixed well by gentle snaking; then incubated at 37?C for 16 hours. After the incubation period, the concentration of SDS was raised to 1% and 0.7 ml. of 'isoamyl alchohol was also added to each solution. After nixing well, equal volur-.es (v/v) of phenol - saturated TE were added. The mixtures were gently nixed for 15 ninutes, then centrifuged at 6000 rpn for 20 minutes. The supernatants were recovered and made 1 t-1 in sodium perchlorate. Equal volumes of chloroform were then added and after mixing well for 10 ninutes, the solutions were centrifuged (6000 rpr.) for 20 ninutes. The supernatants Were recovered and the phenol and chloroform steps repeated until clear

Supernatants were obtained. The final supernatants were made 0.1 M with respect to NaCl and the DNA precipitated by addition of 2 volumes of cold ethanol. The DMAs were recovered on glass rods and dissolved in 30 ml of TE buffer. The precipitation and DMA recovery steps were repeated 3 times and the final recovered DMAs were dissolved in 30 ml of Tris-EDTA. The 0D_n^^nm and Οϋ,,ςθηπι were determined. The ratios OD.,^q/OD_?^ were l.Q.

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-9II. PREPARATION 01' Λ GENOMIC DNA LIBRARY FROM SALMONELLA TYPHIMURIUM

CHROMOSOMAL DNA IN PLASMID VECTOR pNO 1523

II. 1. Sma I AND Hpa'I RESTRICTION ENDONUCLEASE DIGESTIONS

Restriction endonuclease digestions with Sma I (Pharmacia) were macle ·* on 1 Jig of Salmonella typhimurium DNA and 1 jjg of plasmid pNO 1523 DNA in 10 mM Tris-HCl (pH 8.0), 20 mM KC1, 10 mM MgCl₂, 10 mM ME (2-Mercaptoethanol) and 100 jjg (Bovine serum albumin) BSA/ml at 30°C for 2 hours. Digestions with Hpa I restriction endonuclease (Pharmacia) on 1 jig of Salmonella typhimurium DNA and 1 jig of plasmid pNO 1523 DNA were made in 10 mM Tris-Hcl (pH 7-5), 50 mM NaCl, 10 mM MgCl₂ 1 mM ME and BSA lOOjig/ml; at 37°C for 2 hours. The Salmonella typhimurium DNA fragments and the linearized plasmid DNA were tested by electrophoresis on 0.8% (wt/vol) agarose horizontal gels using electrophoretic buffers composed of: 0.04 M Tris-acetate, pH 8.0; 0.002 M Na,, EDTA, 0.01 M NaCl. The electrophoresis was carried out, for 2 hours at 100 volts (Fig. 1).

Ecu Rl and Hin d III restricted lambda phage DN5\ was also loaded onto the gel as a reference. Plasmid pNO 1523 carries both an ampicillin resistance gene (amp^) and a dominant, streptomycin sensitivity gone (StsT^) (fig. 2) which confers streptomycin sensitivity to a streptomycin _s

R resistant (Str ) E. coli MC 1009 host, which carries a streptomycin 5 resistant gene in its chromosomal DNA. It is thought that the Str gene codes for a repressor of the Str^ gone plasmid pNO 15—3 also carries unique Sma I. and Hoa I restriction sites in the dominant Sir' gone.

Insertion of a foreign DNA fragment (eg Salmonella typhimurium DNA fragment) into either of the Sma I or the Hpa I sites of pNO 1523 inactivates S the plasmid Str dominant gene, so no repressor is produced, and effectiveJy restores activit y of the Str^ oh t he host, (E. col i MC 1000) chromosome.

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-10(Fig. 3). This makes it possible to select all transformants carrying recombinant plasmids (ie plasmid pNO 1523 + Salmonella typhimurium DNA fragments) by plating upon media containing both ampicillin and streptomycin. Only those transformant bacteria containing recombinant plasmids would grow on this media. Over 400 transformants were selected on this basis.

II.2. BLUNT END LIGATION

Ligations between Sma I or Hpa I Salmonella typhimurium DNA fragments and linearized plasmid pNO 1523 DNA were carried out using T4 DNA ligase enzyme which is capable of joining DNA fragments with 'sticky ends’ as well as DNA fragments with 'blunt ends'. Ten micrograms (10 jjg) of Sma I or Hpa I digested Salmonella typhimurium DNA fragments were transferred to Eppendorf tubes and 10 jig of Sma I or Hpa I linearized plasmid pNO 1523 DNA, were then added. The following were then added to the reaction mixtures:

jxl H_o0, 10 jil of 10 X ligation buffer (500 mM Tris-Hcl pH 7-S, 50 mM MgCl_o, 10 mM dithiothreiotol, 10 jil of 40(3 polyethylene glycol (P.E.G.) and 10 jjI of T4 DNA ligase. The total reaction volumes were brought to 100 jjI by addition of 20 pi of Il_?0. The reaction mixtures were mixed well and incubated at 15°C for 00 minutes. The reactions were stopped by addition of 0.5 M pH 7·5 Na^ - EDTA to a final concentration of 10 mM.

The ligations were checked by electrophoresis on 0.S% agarose gels with non-ligated DNA as controls. The rest of the ligated materials were divided into small aliquots and stored at -20°C.

II. 3 · BACTERIAL TRANSFORMATION

The host cells (E. col i MC 1000) wen.· pelleted from an overnight LD

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-11culture containing appropriate antibiotics by centrifugation at 5,000.xg for 5 minutes at 4°C. The supernatants were decanted and the pellets were resuspended in 2.5 ml of cold 0.1 M CaCl₂ to render the cells competant for transformation and left on ice for 30 minutes and then centrifuged at 5,000 xg for 5 minutes. More diffuse pellets were observed indicating that the cells had been made competent. The cells were resuspended in 0.2 ml of cold 0.1 M CaCl₂ and transferred to sterile, thin walled glass, tubes for effective heat-shock reaction. E. coli MC 1009 cells were transformed by the addition of O.Ljig recombinant DNA (Sma I or Hpa I cut Salmonella typhimurium DNA fragments ligated with Sma I or Hpa I linearized pNO 1523 plasmid DNA). The reaction mixtures were left on ice for 30 minutes. The reactions were transferred to a 42°C waterbath for 2 minutes and returned briefly to ice. The contents of each of the tubes were transferred to 2 ml of L.B. broth in small flasks and incubated with shaking at. 37°C for 90 minutes. 0.1 ml aliquots of undiluted, 10 , and dilutions were plated onto agar plates containing antibiotics used for selection (.streptomycin and ampicillin) . The plates were incubated at. at 37°C for Io hours, and studied. Λ total of 400 transformants were observed from E. coli MC 1009 bacteria transformed with recombinant Salmonella typhimurium DNA fragments (Sma I or Hpa I digests) and Sma I or Hpa I linearized plasmid pNO 152.3 DNA. All the colonies which grew on plates in the presence of ampicillin and streptomycin were transformants carrying a recombinant plasmid with an insert of Salmonella typhimurium DNA fragment in the Sina T. or Hpa I site which deactivated the

S R

St r gene ot the plasmid pNO 152.)· thus restoring the St r gene in the host bacteria E. col ί MC 1009 chromosomal DNA.

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II.4.

ISOLATION OF PLASMID DNA

11.4.1. RAPID-SMALL SCALE-ALKALINE LYSIS METHOD

Mini cultures (5 ml) were prepared using L.B. medium containing Streptomycin and ampicill in. Each miniculture was innoculated with a single bacterial colony transformant and incubated at 37°C overnight (16 hours). A small volume (1.5 ml) was taken from each culture placed in an Eppendorf tube and the bacterial cells were pelleted by centrifugation for 1 minute in an Eppendorf centrifuge. The remainder of the overnight cultures were stored at 4°C. The culture media was removed by aspiration, leaving the bacterial cell pellets as ary as possible. The pellets were resuspended in IOOjjI of ice-colc solution of 10 mil glucose, 10 r.;M EDTA, 25 t,:M Tris-Cl (pH 8.0) and 4 mg/ml lysozyme, (powder added just before use). The reaction mixtures were incubated at room temperature for 5 minutes before 2C0 jj 1 of freshly prepared, ice-cold solution of 0,2 N NaOH and 1% SOS, was added. The solutions were mixed by inverting the Eppendorf tubes rapidly four times and stored on ice for 5 minutes. An ice-cold solution of 3 11 potassium and 5 M acetate pH 4.8 was prepared and added in the following manner: 60jul, of potassium acetate,

11.5 JU1 Of galcial acetic acid and 23.5 Jll of H^O were mixed and the solution was mixed by gentle shaking in an inverted position and then incubated on ice for 5 minutes. The contents were centrifuged for 5 minutes at 4°C and the supernatants were transfered to fresh tubes after which equal volumes (v/v) of phenol/chloroform were added and the solutions mixed by vortexinn then centrifuged again for 2 minutes. The supernantants from this step were transfered to fresh tubes and the ONA was precipitated by addition of two volur.es of ethanol at room temperature. After mixing by vortexing, the solutions were stored at room temperature for 2 minutes before centrifugation for 5 minutes in an bad original

-13Eppendorf centrifuge at room temperature. The supernatants were removed and the Eppendorf tubes were put in an inverted position on a paper towel to drain out the ethanol. After this step, 1 m.l of 70(5 ethanol was added to each tube and the contents, were mixed for one minute by vortexing before centrifugation for 10 minutes at room i temperature. All the supernatants were removed and the pellets were, dried in a vacuum desiccator or in a Speed Vac. The dried pellets were dissolved in 3θ jil of T.E. (pH 8.0) and DNase free pancreatic RNase (20 jig/ml) was added to each tube and mixed by vortexing. Small aliquots (10 jal) were removed from each isolate and put into a fresh Eppendorf tube. 1.2 jjl of restriction endonucleases Sma I or Hpa I (Pharmacia) were added to each tube and the solutions were incubated for 2 hours at 30°C for Sma I, and 37°C for Hpa I restriction endonucleases. The remainders of the solutions were stored at -20°C until further use. The DNA fragments in the restriction digests, were analysed on 0.5,'S agarose gel (Fig. 4). Non-recombinant pNO 1523 DNA produced one sharp band on the gel (well 3 arrow E). Recombinant pNO 1523 (ie containing inserts of S. typhimurium DNA) produced a sharp band of higher molecular weight (well 4 arrow D). Hin d III digested lambda DNA was used as a reference marker. Commercially available pNO

1523 was also run as a control.

III. SCREENING OF SALMONELLA DNA GENOMIC LIBRARY

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The screening of the Salmonella typbimurium DNA genomic library in order to identify a Salmonella - specific recombinant DNA molecule was divided into two parts; (i) the primary screening stages and (ii) the second screening stage's. In the primary screening stage, plasmid

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DNA was isolated from colonies of bacteria transformed with recombinant plasmid (pNO 1523 + Salmonella DNA). In order to isolate plasmid DNA from a large number of colonies, the rapid - small-scale DNA isolation method was used which enabled the handling of 36 colonies at 'a time . The plasmid DNAs thus prepared were tested on 0.8% agarose gels using the normal non-recombinant plasmids as controls (Fig. 5)· All plasmids which indicated higher molecular weights by their electrophoretic mobilities were rescreened (eg Nos.

41, 43» 46, 50 etc). It was found necessary to divide the gel slabs into two equal,portions by using two gel-combs, one at the top and one in the middle of the gel. This enabled the use of one slab-gel to run a large number of samples in order to screen the over 400 colony transformants in as short a time as possible. This enabled us to screen out recombinant plasmids. We screened a total of 400 colonies in the shortest possible time (3 weeks). These were colonies from bacterial transformants carrying the Salmonella recombinant DNA in plasmid pNO 152.3 prepared as described above.

The selection of recombinant plasmids carrying Salmond 1 a typhimurium DNA inserts which were Salmonel la-specific in the second screening stage, was then achieved using E. coli and Salmonella chromosomal DNAs which had been radioactively labelled with radioisotope, using the nick translation procedure. The selected recombinant plasmids were digested with restriction endonucleases Hpa I and Sma I, the same endonucleases which had been used in making the fragments for ligation experiments, to separate pNO 1523 DNA from the cloned S typhimurium DNA fragments. The DNA fragments were transferred to nitrocellulose using the Southern Blot procedure.

The Salmonella - specific fragments were selected by hybridization of the DNA fragments .on nitrocellulose, with a primary E. coli chromosomal DNA probe, using a Salmonella typhimurium DNA probe as . ________ bad original

-15control (Fig. 7)· All those fragments which cross-hybridized with

E. coli chromosomal DNA were eliminated. Only those fragments which were Salmond la - specific and did not hybridize with E. coli chromosomal DNA, were selected for the secondary screening. E. coli chromosomal DNA was thus used as a primary screening probe because it was believed that the presence of E. coli DNA sequences, would be universal.

It was necessary to have a Salmond la-specific DNA probe which would not τϊcross-hybridize with E. coli contaminants. Eight (8) clones which did not hybridize with E. coli DNA were selected for the secondary screening. These were Nos. 3, 8, 12, ,13, 15, 32, 35 and 44·

III.l. PRIMARY SCREENING BY AGAROSE GEL ELECTROPHORESIS

In order to screen the greater than 400 transformants with recombinant plasmids carrying Salmonella typhimurium DNA inserts of varying sizes as rapidly and as efficiently as possible, a method which enabled iis to run a large number of samples on one gd was developed. In this method, a slab 0.8% agarose gd 24 x I8 cm was poured and two gd combs were used, one placed at the top of the gel and another in the centre of the gd to give two equal gel portions between the walls. After the agarose gd had set, as many as 36 different samples of isolated and purified plasmid DNA could be run * on the same gd. and hybrid plasmids were recognised by virtue of their higher molecule weights depicted by slower mobilities on the gel compared to non-recombinant plasmid pNO 1523 (Fig· 6). The position of the linearized pNO 1523 DNA is shown in well 2 (pNO + Hpa I). All other posit ions show 5. tyn’n imudnm DMA fragments, either smaller or larger than the linearized plasmid DNA. Recombinant plasmids were select,ed on this basis for further screening by DNA-DNA hybridization.

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-16Using the results presented in Fig. 7, the following colonies were selected as possessing hybrid plasmids carrying Salmonella typhimurium DNA inserts: Nos. 4b 43, 46, 50, 57, §0 and 8?. All 400 transformants were screened in this manner and colonies which had been shown to possess recombinant plasmids were selected and subjected to a second stage of primary screening which involved DNA-DNA hybridization using radioactive labelled Salmonella typhimurium chromosomal DNA as a positive control probe and E. coli chromosomal DNA as an experimental probe.

Plasmid DNA. was isolated using the Rapid-Small-Scale method, (Holmes & Quiahy, 1CS1) and the purified recombinant plasmid DNA was digested with restriction endonucleases Hpa I or Sma I in order to excise the inserted Salmonella typhimurium DNA fragments. The excised Salmonella typhimurium DNA fragments were separated from the linearized plasmid DNAs by electrophoresis on 0,2% agarose gel divided into two equal portions as described above. The digests were loaded on to the gel slots in duplicates in sections A and B of the divided gels in such a way that equal amounts (1 jug DNA of No. 29, 30, etc.) were loaded on A and B sections. After electrophoresis the cels were stained with ethidium bromide and photographed (Figs. 6). The DNAs were immobilized and filter-bound onto Nitrocellulose filter by the Southern Blot method.

The filters were then cut into two sections A and B. Section A was hybridized with Salmonel la typhimurium chromosomal DNA used as a 32 positive control, and radiolabelled with P-d-CTP following the nick translation method (Maniatis, Jeffrey, and Kleid, 1975 ; Rigby, Dieckmann; Rhodes and Eerg; 1977; Mathew, 1923, and Anershar.i 1920). The B section of the filters was hybridized with £. coli chromosomal DNA ►radiolabelled with -^P-d-ATP and -l^P-d-ACT as above. Tin? _______

BAD ORIGINAL hybridized DMAs on filters were processed for autoradiography and analysed for presence of Salmonella-specific inserts. The excised Salmonella typhimurium DNA inserts which did not hybridize with _E. col i chromosomal DNA v/ere selected as semi-Salmonel la-specific clones (Fie.

7) Eight Salmonella typhimurium DNA fragments from colonies, 3, 8, 12,

12, 15, 32, 35 and 44 v/ere selected using this method. These were thin processed for secondary screening.

111.2. SOUTHERN BLOTS

After the gels had been stained with ethidium bromide and. photographed, they v/ere trimmed to remove the unused areas. The gels were soaked in 1,5 H NaCl, and 0,5 H NaOH for 30 minutes at room temperature to denature the DNA. The cels were then washed in H_o0 (distilled), for 5 minutes then neutralized by soaking in three 30 minutes changes in 0.5 H Tris-Hcl, pH 7, and 3 II NaCl at room temperature with constant stirring. After the last soaking, the gels were soaked in 20 x SSC (3 II NaCl, 0,3 H sodium citrate) pH 7· for one hour. Nitrocellulose filters v/ere cut so that they were 3 mm smaller than the gels in both dimensions. Also 2 pieces of 3 HU Whatman paper per gel, v/ere cut 3 ram smaller than the _ nitrocellulose filters in both dimensions and lastly a pile of 3 mm filter paper was cut 3 ra smaller than the 3 Ufl Whatman filter paper. A piece of Whatman 3 UH paper (33 cm x 28 cm) was wrapped around each piece of plexiglass and both pieces were placed in the middle of large baking dishes and the dishes v/ere filled with 20 x SSC nearly to the top of the plexiglass support. The Whatman papers around the plexiglasses had all the 4 overlapping sides soaking in the 20 x SSC to form a wick. Air bubbles between the plexiglasses and the Whatman paper wick were carefully ____________________________________ BAD ORIGINAL

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-18removed. The gels were carefully placed on the damp 3 Mil Whatman papers and air bubbles removed. The Nitrocellulose filters were marked in a pan and then soaked in distilled H/0 for 2 minutes and then in 20 x SSC for 10 minutes, and then were placed on the gels and ail air bubbles removed before two sheets of damp 3 ίίίί Whatman papers were placed on each. Air bubbles v/ere removed carefully, then piles of Whatman filter papers were placed on each one of tlie systems to a height of 10 cm. Glassplates were placed on the 10 cm stack of papers before weights of 500 g were finally placed on top. Transfers were allowed to proceed for 24 hcurs. The filter papers and the 3 MM filters above the gels v/ere removed and then the nitrocellulose filters were also removed and rinsed in 3 x SSc for 5 minutes. The filters were blotted betv/sen two 3 MM Whatman papers and dried at ,37°C for one hour. The gels were restained in .ethidium bromide and rephotographed to check the transfers. The dried nitrocellulose filters were placed between two sheets of 3 MM Whatman papers and then placed between two glass plastes and baked for 2 hours at · 8O°C.

111.3. NICK TRANSLATION

The Sal monel la typhinuriur.i DNA genomic library was screened using £. co 1 i HC 600 chromosomal DflA as an experimental screening probe, while Salmonella typhimuriuni chromosomal DNA was used as a control probe. Any Sal monel la clones which did not^- hybridize with E. col i MC'600 labelled probe were considered to be

Salmond la specific clones. For these experiments, both E. col i I'O

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-19600 chromosomal DNA and Sal none! la typh imur iur, chromosomal DNA were radioactively labelled with radiosotope 32P using the nick translation proceedure. For the nick translation reaction mixtures, two Eppendorf tubes were taken. One was marked (Salmonella) and the other £. (£. coli) and 0.5 and 0.5 to 1 jug of respective DNA was added to each tube. The following were added to each reaction mixture:

2.5 JjI. of 10 x nick translation buffer (50 mil ixgCl^, 500_pg/ml BSA and G.5 li Tris-HCl, pH 7,5), 2 ul-0,2 mii-d GTP, 0,2 mfl-d TTP,4jj1 ³²p d-CTP (400-2000 ci/ml mol: CCil Ci/ml); 4 ul ³²P - d ATP (400-20C0 Ci/m l-icl: 10 mCi/ml): freshly prepared from 2 mg/nil of stock solution stored at - 20^cC) and lj.il DNA polymerase 1 (5 units). The reaction mixtures were incubated at 16°C for 2 hours and the reaction mixtures were stopped by additon of 2.5 jjI of 0.5 H EDTA. The unincorporated radicnucleotides were separated from the radioactively labelled DNA on a G 50 sephadex chromatography column, Pasteur pipettes were plugged with glass wool, siliconised and columns of sephadex G 50 were poured and equilibrated with several volumes of TE buffer. The DNA which is excluded from the sephadex matrix and elutes ahead of the unincoorporated deoxyribonucleotides was collected in 200jul fractions and counted directly by Cerenhov counting. The fractions.containing the DNA were pooled together and stored at 4°C.

AP 0 0 0 0 9 fi

III.4. HYBRIDIZATION OF FILTER - BOUND DNA

The nitrocellulose filters were prepared for hybridization by soaking ‘the baked filters in 3 x SSC (0.45 M NaCl, C.045 fl trisodium citrate) buffer for 10 minutes. The filters were then transferred to hybridization boxes containing prehybridization solution (3 x SSC, O.Ki SDS, 5 x Denhardt's solution (50 X Denhardts/2« BSA, 2% Ficoll and 2% polyvinyIpyrrolidore. Stored at -2U°C) and ICO jjg/ml of denatured______

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Salmon Spem D'lA). The filters were incubated in the prehybridisation solutions at 65°C for 4 hours. The prehybridization solutions were removed after 4 hours and replaced with hybridisation buffers (3 x SSC, 0.01 M EDTA), 3-f? labelled DilA probes which were denatured by incubation at 1OO°C for 10 minutes, 5 x Denhardt's .solution, 0.1%,SDS and 100 jag/ml denatured salmon sperm DilA). The hybridisation solutions, in boxes, were incubated at 65°C for 40 hours. At the end of the hybricisation period, the hybridisation solutions were removed and stored to await the hybridisation experiment results. The filters were washed with low stringency buffers (3 x SSC,' 0.1% SDS) for .30 minutes with constant agitation at 65°C. An average of 3 washes at low stringency conditions and two high stringency, conditions (l x SSC, 0.1% SDS) were carried out on each set of filters.

The level of radioactivity was checked at the end of each wash. One more higher stringency condition (G.l x SSC, 0.5% SDS) wash was made if the levels of radioactivity were found tc be high. When the level of radioactivity was reduced to acceptable levels (to ensure a lew level of background radioactivity), the filters were removed and dried by placing them between two 3 Mil Whatman filter papers and incubating at 37°C for one hour to dry. After the 'filters were dry, they were transferred to autoradiography conditions. The hybridizations were analysed by examining the autoradiograms (Fig. 7).

IV. 'SECONDARY SCREEilliiG BY COLONY HYBRIDIZATION

Plasmid DilA was isolated from the eight colonies which 'were selected for the secondary level screening, using the large-scale plasmid DilA isolation method (Godson and Vapnek 1S73). The recombinant DilA clones from each colony were· purified by separating them from the plasmid DilA by low melting temperature gel electrophoresis following restriction _____.

___------------------ 01

-21endonuclease digestions with Hpa I or-Sma, I. The separated fragments were cut and recovered from the LTil-gel (Smith, 1530; Chen and Thomas. 1530) (Fig. 3). The purified recominant Salmonel la DNA fragments were labelled with radioisotopes ^²P-d-ATP and 3-p_d-CTP following the nick-translation method and were then used as probes in colony hybridization experiments to check specificity for Salmonellae and possibility of cross-reaction with a wide range of other enterobacteriaceae.

IV. 1. LARGE SCALE PLASMID DNA PREPARATION

Plamid DNAs from the selected eigbt colonies (colony numbers 3, 8,

12, 13, 15, 32, 35 and 44) were isolated following the large-scale plasmid DNA preparation methods. Precultures were made by inoculating 15 ml L.S. cultures (containing the antibiotics ampicillin and screploymyin) with single bacterial colony transformants generated by ligating Sma I or Hpa I linearized Salmonella typhimurium DNA fragments with Sma I or Hpa I linearized plasmid pNO 1523 DNA. 5 ml were taken from each preculture ana used to inoculate one litre L3 cultures with antibiotics amplicillin (50 jug/nl) and streptomycin (25jjg/ml). The liquid cultures were incubated at 37°C with shaking until the ΟΟ^θ'reached O.S or 1.

Chloramphenicol (34jua/ml) was added to a final concentration of 170jig/ml and the cultures were incubated at 37°C for lo hours to allow plasmid amplification. The cells were harvested by centrifugation at 5000 rpm for 10 minutes at 4°C. The pellets were resuspended in 5 ml of 25% sucrose, 50 mil Tris-HCl, and 1 mil EDTA (pH 3.0). 5 ml of lysozyme (10 jug/ml in H^O) were added and the solutions were mixed well and stored on ice for 10 minutes before 5 ml of 0.25 Nt EDTA, pH 8.5 was added on ice. The solutions were mixed on ice for 10 minutes after which 35 ml of 0.1% Trutib x 100, 0.5 mil-Tris-HC! (pH 8) and 50 mii EDTA were added. The mixtures were shaken gently and incubated on ice for one hour.___......

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ΑΡ00009Θ

-22Centrifugation of the lysates at 18000 rpm for one hour was carried out at 4°C. The supernatant-v/as recovered and the volun.es carefully · measured after which 1/4 (one quarter) of the original volume of supernatant (v/v) of 5 1-1 NaCl was added to the supernatant followed by the addition of 1/3 volume (v/v) of the original supernatant, of 10 mil Tris-h’Cl (pH S.O), 1 r.:ll EDTA and 40’/ P.E.G. 6000. The solutions were at this point incubated at 4°C overnight (at least 16 hours). The preparations were then centrifuged at 10 000 rpn for 20 minutes at 4°C. Each pellet (precipitate) was resuspended in 10 ml of TE (pH 8) buffer and prepared for ultracentrifugation of CsCl and ethidium bromide. · (6.2 ml of each solution + 7.1 gm CsCl '+ O.S ml. ethidium bromide (10jjg/ml in HC1). The preparations v/ere mixed well to make sure all the CsCl was well dissolved and centrifuged at 10 OCC rpm for 30·minutes at 4°C to remove proteins which formed a complex with ethidium bromide appearing as a red layer on the top of the centrifuge tubes. The rest of the solutions were transferee to fresh pclyamyl tubes and centrifuged at 40 0( hours at 25°C. At the end of the run, both the chromosomal and plasmid DiiA bands were located with U.V. light (Long Lave) and the plasmid DNA band was recovered. Ethidium bromide was removed by adding an equal volume of 5 I·; HaCl in T.E. saturated isopropylalconol (100 ml 5 M f!aC 1 in

T.E. + 100 'r.il Isopropanol), followed by mixing, with shaking and centrifuging at 1500 xg for 3 minutes at room temperature. The lower aqueous phases were transferred to clean glass tubes and the extractions were repeated 4 to 6 times until the pink colour dissappeared from tiie aqueous solutions. The aqueous solutions were dialysed against 4 changes of T.E. buffer pH 8.0 and the preparations were analysed on 0.8/ agarose gels (Fig. 6).

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-23IV.2. EXCISION OF SALMONELLA TY PH I HUP. I UH Di.'A INSERTS FROM PLASMID DNA BY ENDONUCLEASE DIGESTION

After the purification of plasmid DNA fror.i colony 15 which carried a Salmonella fragment insert (clone 15) it was necessary to excise the fragment and separate it from the plasnid pNO.1523 DNA in order to latei the fragment of Salmonella typhimuriur chromosomal DiiA, to be used as a Salmonella-specific DNA probe. This involved two important steps. First, it was necessary to use the same restriction endonuclease enzyme Hpa I which had been used in generating the Salmond la typhimuriun DNA’ genomic library, in order to excise the cloned Salmonella typhimuriur.;

DNA fragment. The excised fragment was separated from the resultant linearized plasmid pNO 1523 DNA on a low melting temperature .agarose gel and recovered by cutting the fragment band revealed on Ultraviolet (Long wave) radiation after staining with ethidium bromide. The DNA fragment, thus recovered, was purified using phenol-chleroform extraction.

860000dV

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X

-24IV.3. RESTRICTION ENDONUCLEASE Hpa I DIGESTION TO EXCISE CLONE 15 FROM PLASMID pNO 1523 DNA.

The reaction mixtures

Salmonella typhimurium DNA as outlined below; using E for the Hpa I endonuclease digestion to excise from clone 15 from plasmid pNO 1523 were set up coli 600 chromosomal DNA as control.

Tube uo ·	Sample Type	Amount of DNA	Digestion Buffer	Disti 1 led Water	Hpa I Enzyme	Total Volume
1	Plasr.id DNA-Carryin Clone 15	r 40 jug	SO J.1	23 jul	4 jj!	SO jjl
2	E. coli	10 jug	1.2 j;l	8.0 jtl	l.Ojd	20 fd.

The reaction mixtures were incubated at 37°C for 2 hours and the reactions were stopped by the addition of 10 mM EDTA pH £.0 (final concentration) The digests were tested by gel-electrophoresis for 2 hours, on C.o agarose gel.

IV.4. SEPARATION AND RECOVERY OF CLONE 15 FROM A LOW MELTING TEMPERATURE

AGAROSE GEL.

A C-6% low melt ing temperature gel was prepared by mixing 0. 6 g of low temperature melting agarose gel in 100 ml of TAE buffer and after melting the gel, 30 jjI of ethidium bromide (10 mg/ml) was added to the agarose gel solution. The mixture was poured onto the slab gel form and bad original left to set for 2 hours at 4°C before use. Special gel combs were used to cut bigger wells into the gels. After the gels had set well, the digested-hybrid plasmid from colony 15, were loaded into one big well.

The gel was run at 80 volts by 80 millconbs per hour for 16 hours (overnight). The clone 15 (S.t 1 rrone 11 a fragment) DNA band was localized with UV light long wave at the end of the run. Pieces of the low · temperature melting gel containing the clone 15 fragments separated from the linearized plasmid pNO 1523 DNA, were cut out, using a sharp razor blade. The gel pieces were placed in glass centrifuge sterile.tubes and the gels were melted by incubation at 65°C for 10 minutes. Equal volumes of phenol were added and the mixtures were centrifuged for £ minutes at 700 ,'rpm to separate the agarose from the DNA fragments. The aqueous phases containing the DNA fragments were transfered to clean sterile Eppendorf tubes. Equal volumes of phenol/chloroform (50v/50v) were added and the resultant solutions were mixed well before centrifugation for Ί5 minutes in a microfuge. The aqueous phases were again transfered to clean sterile Eppendorf tubes and equal volumes of chloroform isoamyl alchohol (24.1 v/v) were added. After a good mixing, the solutions were centrifuged for 15 minutes in a microfuge at maximum speed. The aqueous phases from this step were transfered to clean Eppendorf tubes before the DNA ’was precipitated by the addition of two volumes of cold ethanol followed by incubation at -30°C overnight.

The precipitated DfJA was collected by centrifugation in microfuge for 20 minutes. The pelleted DNAs were air dried before they were resuspended in 10 HM Tris-EDTA buffer pH 8.0. The recovered DNA clone 15 fragments were tested on 0,8% agarose gel (Fig. S). The concentration of the recovered clone 3 5 fragment DNA was determined by UV spectrophotometry and stored at -30°C for use as a Salmonella - specific DNA probe.

The Salmonella typhimuriun DNA fragment from clone 15 was radioisotopically labelled with ^P-d-ATP and 3“ P-d-CTP following bad original A . AP0 0 0 0 9 8

-26the nick-translation r.iethod as described above, to be used as a probe for the detection of Sal monel la in foods, and veterinary and clinical samples.

IV.5. COLONY HYBRIDIZATION: LYSIS AND PREPARATION CF FILTER BOUND' DNA

For colony hybridization, master plates were prepared, by spiking with a toothpick acar plates divided into 24 appropriate places and placing 24 bacterial colonies. The plates were incubated at 37°C until the colonies ( ca 0.5mm.) were visible. Dry, sterile nitrocellulose filters, cut to appropriate sizes, were labelled with a soft pencil, and were placed carefully on the surfaces of respective master plates with the ; numbered sides in contact with the colonies, being careful to ensure that the contact was made over the whole surface, without air bubbles, and that the filters were completely uniformly moistured. Each filter was marked. The filters were then transferred, colony side uppermost, to fresh agar plates containing appropriate antibiotics, where applicable, and incubated at 37°C for several hours to allow the colonies to grow-. At the same time the master plates were also incubated at 37°C for a few hours before'storage at. 4°C. After the colonies had grown on the filters, the filters were transfered onto pads of filter paper pre-soaked in 0.5 M sodium hydroxice, colony' side uppermost, making sure that no air bubbles were trapped underneath the filter membranes. This step lysed the bacterial colonies and denatured their DNA. Care was taken not to allow any liquid to flow onto the top surface of the filter membrances. After 10 minutes of lysing in this manner, the membranes were transfered to pads of 3 Mil Whatman filter papers pre-soaked in 1 H Tris-HCl, pH 7.4 and incubated for 4 minutes. The membranes were then transfered to 3 I’M paper pre-soaked in 0.5 M Na-Cl and 1 H Tris-HCl pH 7.4 buffer. The filters were incubated in this buffer system for at least 15 minutes b£fore_be.ing.

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-27rer.ioved and air dried. The air dried filters were baked in a vacuum oven at 80°C for 2 hours. The filters at this stage contained filter bound denatured DNAs imprinted in the patterns of the colonies on the original master plates. The filter bound DNAs were prepared and hybridized using the Salmonella typhinuriurn. DNA clones radiolabelled with 32p_d-ATP and ³²P'd-CTP as probes, following the nicktranslation method. Initially Salmonella typhimurium DNA clones 3, 8,

12, 13, 15, 32, 35 and 44 were used as probes and clone 15 was found to be Salmonella-specific in that it hybridized with all Sal monellae tested and was negative against all other enterobacteriaceae tested (Fig. 9).

IV.6. FURTHER COLONY HYBRIDIZATION TESTS o

After establishing clone 15 to be a Salmonella - specific probe, the length of the Hpa I Sahnonelda typhimurium DNA fragment from clone 15 was determined by 0.8% agarose gel electrophoresis in comparison to Hin d III digested lambda DNA to be 2.35 kilobase pairs (Fig. 10). Clone 15 was radioisotopically labelled as described before and used as a probe for further colony hybridization tests with 5b Salmonella strains (Collection de l'lnstitut Pasteur C.I.P.) and 50 Salmonella strains located from meat and meat products, from Centre Technique de la Salalson de la Charcuterie et des Conserves de Viandes. Maisons-Alfort-, France and from 32 Salmonella strains isolated from meat and meat products identified by the Meat Hygiene Division of the Department of Veterinary Services in the Ministry of Agriculture, Salmonella Typing Laboratory in Zimbabwe.

Minicultures were prepared in 5 ml of L.B. broth as described above for each of the lyophilized bacteria received from the Pasteur Institute Collection (CIP) and^from the Meat Hygiene Laboratory (Centre Technique de la Salaison de la Charcuterie et des Conserves de Viandes,

Maisons-Al fort., France). The bacterial cells were streaked on agar _________________ ____________________BADORIGINAL

AP 0 0 A A o

-2δplates and after 15 hours, master plates v/ere prepared on agar plates in a special numerical order. The bacteria v/ere transfered onto a nitrocellulose filter by the replica plating method. Disks of nitrocellulose filters were cut to the same measurements of the petri dish sizes on which the master plates were made. The nitrocellulose filters were carefully overlaid on the bacterial master plates, being careful to wet the filter evenly and also being careful not to allow any air bubbles to be trapped between the nitrocellulose filters and the bacterial master colonies on the agar plates. The nitrocellulose filters v/ere peeled off carefully from the master plates and transferred to another agar plate, colony-side up and incubated at 37°C for an hour or until the colonies had grown well on the filter. The cells on nitrocellulose filters were lysed and prepared for DNA-DfiA hybridization as described above.

IV.7 TEST CF PROSE SENSITIVITY

In most cases of Sail-·,one! la infection, such as diarrhoea, enteric fevers and food poisoning, Sal morel la is found in the presence of competing enterobacteriaceae which cause similar types of disease symptoms. In such cases, it is not possible to detect presence of Salmonella in toe presence of competitive bacteria using the classical microbiological method. It is only possible to detect the presence of one Salmonel la cell in cases where the sample being tested did not have competing bacteria (Edel and Kampelnacher, 1269). When large numbers of competitive bacteria are present, only about two thirds of the actual samples tested may be identified. Tin's means that in such a competitive situation, about tv/o thirds of the bacteria in the heterogeneous population must be Salmonella in order to get positive detection using the classical microbiological method. The need for a more sensitive method cannot

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-29be overemphasized. The degree of sensitivity of the Salmonella specific DNA probe of the invention was established by dot-blot hybridization experiments using heterogeneous populations of bacteria. Salmonella typhimurium (10^ cells/ml) was spotted on nitrocellulose in a total volume of 20 jul (spot C in Fig. 11). In spot A', 10^ cells/ml of Salmonella typhimurium and 10^J cells/ml of Shigella were mixed, pelleted by centrifugation and cells resuspended in a total volume of 20 jal. The whole 20 jil volume was spotted on mitrocellulose spot A. In spot B only 10^J cells/ml of Salmonella typhimurium and 10^ cells/ml Shigella were spotted. The bacterial cells thus spotted, were lysed and hybridized with the Salmonella specific probe as described above and as shown in Fig. 11.

In a similar experiment, a serial dilution of Salmonella typhimurium (pure culture) was made to give 10^/cells/ml, (undiluted) 10^ cells/ml, 10^'^ cells/ml and ΙΟθ’?^ cells/ml. The bacterial cells were pelleted by centrifugation and resuspended in total volumes of 20 jal and spotted on a nitrocellulose filter as shown in figure 12 (A-D). In the second part of this experiment,serial dilutions of Salmonella typhimurium made above were mixed with several competing enterobacteriaceae. The cell concentrations of Shigella. Citrobacter

APO 0 0 0 9 8 f reundi ,. E. coli, Serratia liquefaciens, Klebsiella ozonea were kept at 10^/cell/ml to give a mixture of 10^ cells/ml S. typhimurium + 10^ cells/ml Shigella + 10^ cells/ml Citrobacter freundi .+ 10^ cells/ml

E. coli + 10 cells/ml Serratia liquefaciens + 10^u cells/ml Klebsiella ozonea on spot E. For spots F-I, the same concentrations of competing bacteria were kept at 10^ cells/ml, while the concentration of S.

IS typhimurium were 10^J cells/ml on spot F, 10 cells/ml for spot G,

0.7 5 , cells/ml for spots H and I.

bad original (

*ί

-30USE OF THE SALMONELLA-SPECIFIC DNA PROBE IN ROUTINE SAMPLING OF

SALMONELLA IN MEAT AND MEAT PRODUCTS AND CLINICAL DIARRHOEAL CASES.

One thousand (1000) samples were received from the Meat Hygiene Division of the Department of Veterinary Services in Zimbabwe and at the same time 4θ0 samples came from the City Health Department, Public Health Laboratory at Bulawayo, Mpilo General Hospital and Parercnyatwa General Hospital Harare. All the above samples which had been previously tested by the conventional method, were received after incubation in selenite liquid broth for 24 hours at 37°C and tested using the Salmonella-specific DNA probe of the invention.

The bacterial cells were pelleted by centrifugation in a microfuge. The pelleted cells were washed in distilled water and resuspended in 20 ul volumes of distilled water. The 20 jil cell suspensions were spotted on nitrocellulose filters (20jjl/spot) to form dot-blots for DNA-DNA hybridization experiments. The filter spotted bacterial blots were lysed as described above, to filter bind and denature DNA for DNA-DNA hybridization with the Salmonella specific DNA probe which had been radio-labelled with 32p-d-ATP and 32p_d-CTP following the nick translation procedure described above.

The results are shown in Fig. 13.

Samples 1 to 40 represent part of' the 1400 samples tested.

Results were scored as +++ for more than 50% presence, ++ for 20-40½ and + for 1 to 20%. In Fig. 13 numbers 5, 7, 11 and 34 would represent samples with a heterogeneous population of bacteria with more than 50% Salmonella. Numbers 2, 30 and 37 represent heterogeneous populat ion of bact eria with less than 30% Salmonella. while numbers 14, 19, 25, 26, 35, 36 and 40 represent samples in which less than 20% Salmonella is present in a sample of heterogeneous ** population of competing bacteria. In (.his particular case, only ._____—----------- BAD ORIGINAL

-31samples 5, 7 and 11 had been detected as positive for Salmonellae using the conventional method. All the rest (Nos. 2, 14, 19, 24, 26, 30, 34, 35, 36, 37, 3S and 40), had been diagnosed as negative for Salmonella presence.

When the probe from clone 15 was tested by DNA-DNA hybridization, it hybridized with all the 56 Salmonella strains from the collection of Pasteur Institute (CIP) (Table 1). When 5θ representative species of enterobacteriaceae-isolated from meat and meat-by products, were used as negative controls in DNA-DNA hybridization, the probe was negative against all the 5θ negative controls. This demonstrated the probes specificity for Salmonellae .(Table 2Λ and B).

When Salmonella - specific DNA probe was further tested against 32 Salmonella strains provided by the Meat Hygiene Division of the Department of Veterinary Services in the Ministry of Agriculture, Zimbabwe, all the samples showed positive hybridization results (Table 3).

ROUTINE DIAGNOSIS 0E SALMONELLAE SPP.

APO00098

When the probe was used in screening for Salmonella presence in 300 samples from meat and meat products, 200 swabs samples from a number of sites and materials taken from the main cattle, sheep and pig abattoirs, 500 rectal swabs samples from abattoir employees from the Meat Hygiene Division of the Department of Veterinary Services, Ministry of Agriculture, Zimbabwe and 400 clinical diarrhoeal samples from general hospitals and

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Public Health Laboratories, in Zimbabwe, previously tested with the conventional method, the Salr.rencl la-specific DNA probe was found to be more specific, more sensitive and more cost effective in detectirc presence of Salmonellae in the presence of competitive heterogenous populations of bacteria. In clinical diarrhoea! cases 63.9% of the samples which had been diagnosed as negative for presence of Salmonellae using the conventional method were diagnosed as positive using the Df.'A probe method. In rectal swabs of abattoir employees 43.9% more positives were detected with the probe method. When 200 swabs samples taken from various sites and materials from the main cattle, sheep and pig abattoirs, in- Zimbabwe were tested, 41,.7% more samples were detected as positives.

In the case of 150 meat and bone meal samples from the meat products, 43.4% more positives were detected with the probe method and for the 150 blood meal samples tested, 36.4% more positives were detected. The total number of samples tested by th.e two methods is 1400 and 52% of these (736 ) were detected positive for the presence of Sal monel la using tne probe method while only 3.71% (52) of the same 1400 samples had been detected positive for Sal monel la presence using the conventional microbiological methods. These results are summarized on Table 4.

COST-EFFECTIVENESS

When a comparative study was made to determine the cost effectiveness of the two methods with respect to time and materials needed, the prcce method came out far more cost effective than the conventional method. For the conventional method, samples were brought in the laboratory after 2hour incubation at 37°C in selenite broth. The samples were then transferred to selenite-lactose enrichment broth, or tetrathionate brctr. and further incubated for 24 hours at 37°C. Aftr tins incubation, a few drops were streaked onto selective media (-lactose-sucrose-pjyenc; _________________- BAD ORIGINAL

-33agar) and incubated for another 24 hours. Suspect colonies from this step were subinoculated by stabbing and streaking a butt/slant each of Kligler iron-lactose dextrose-phenol red, lysin iron-dextrose phenol rod agar.

This was further incubated for 24 hrs at 37°C. Suspected cultures at this stage were tested against polyvalent 0 and H (Welcome Ltd) antisera for IS hours and then submitted for typing to the Heat Hygiene Division Salmonella typing Laboratory. The conventional method requires at least 114 hrs to give some sort of indication of the presence of Salmonel la in any sample.

Detection of Salmonella presence in any sample brought to our laboratory in a 24 hour selenite non-seletive culture brctii requires only 24 hrs to obtain specific, sensitive reliable and definitive results. The bacterial cells are pelletted, spotted onto nitrocellulose filter (1C minutes) and lysed to denature and filter bind the DHA (2 hrs) by baking trie filters at cG°C. The prepared filters are processed by hybridization v.'ith probe DilA previuusiy racioiSotopically labelled v.itii 32P-d-ATP and32P-d-CTP using nick translation (12 hrs). The hybridization is detected by autoradiography which takes about 6 hrs incubation dependent on signal strength. The total cost investment is in the buying of.the proce Kit, nitrocellulose filters Xray films and developing chemicals. The procedure is time effective, cost effective, and simple to operate. It does not involve a lot of expensive chemica]s and special selective media anc indicator chemicals required by the conventional met hods ot’ detecting Saltnonelae spp. ·

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P		P	P
P	£2	P	to
	c	03	03
r-	P	—1	—J
bi	g	bi	bi
£		£	P
P	•H	O	0
JO	u	Pj			>·.
c	-J	c	c	03
P	E	P	to	c	r-
p	•M	P	p	ip
¢4-,	P	P	u-		0
c	o.	c	c	£
0		P	0	c	03
7)	P	73	f)	•r-4
ce	ce	ce	m	ce	re
CM	LO			— =0
CO	CM	o	co	lo
o	r—		co	CM CM
^3-	<r	*3-	*3

LO

CM ^Γ ^rn

Ο Γ- LD

C** ο CM

CM CM CM xr tn tr

CM

τ.

cr>

vo

CM

P»

CM

					4-
					LT;
					—		c
							I
					E		•H
	3				43 TO	0J	u
c	G	re	c		£	c	E
•—4	P	E	P	c	P	P
r—4	c	T	TO	P	4->	N	P
P		c	c	•H	yj	♦r^	CL
—	ij	o:	P	f	E	P
to	71	c.	t—4	P	03	03	P
re	re	re	ω	in	ce	re	re
o	r^.	LO	co	’40	o	o	CM
co	co	^3-	CM	C	LO	CM	LO
CM	CM	CM	r—		r—	in	CJ
	*3	<3·	*3“			LO

Table cent.

BAD ORIGINAL ft a

TABLE 2A

-36EflTEROCACTERIACEAE, PASTEUR INSTITUTE COLLECTION - TESTED AGAINST

SALMONELLA.

SEROTYPE NUliCER	BACTERIAL SPECIES	REACTION RESULT +/-
55.7	Citrobacter freundi
MC 1009	E. coli	-
7211	Levinia malonatica	-
Ent. 75	Enterobacter doacae	-
A 253	Providincia	-
5473	Shigella boydi	-
6215	Edwardsiella lardi	-
A 250	Enterobacter hofnia
Ent. 65	Enterobacter agglomeransy
A 237	Pseudomonos aerugenosa	-
3327	Yersinia enterocolitica	-
E 62	E. coli	-
SM 13	Sei’ratia sp.	-
55213	Klebsiella ozonea	-
En 65	Enterobacter aerogenes
6916	Proteus Vulagris
pr 11	Proteus microbilis
pr 14	Proteus Vulgaris	-
• 5256	Serratia liquefaciens	-
A 253	P rov i dene ia ale ale f ec ions
os 523	Pseudomonas aerugeni osa
*		J

TABLE 2E -37ENTERCBACTERIGACEAE ISOLATED FROF FEAT AND I TEAT PRODUCTS (CENTRE TECHNIQUE DE LA CHARCUTERIE ET DES CONSERVES DE VTANDES MAISONS ALFORT FRANCE. ENTEROGACTERIACEAE AGAINST T.'liI CH SALMONELLA SPECIFIC DNA PRCCE LAS TESTED EY DNA DNA COLONY HYBRIDIZATION.

SERO-	BACTERIAL SPECIES	RESULT	SERG-	BACTERIAL SPECIES	RESULT
TYPE		+/-	TYPE		+/-
KUFGEP.		•	NNUFEER		- .

Ci 24	Citrobactor freundi		SF	10
Ci 23	C. freundi	-	Rl	74
Ci 24	C. freundi	-	Rio	37
Ci 27	C. freundi	-	fc	ICG J
E51	E. coli	. -	fc	SCO
E 20	E. coli	-
E57	E. coli	-	Pr	14
E62	E. coli	-	Eu	75
EulCC	E. coli	-	El	76
Eu23	Enterobacter cloaceae	-	Pr	81
Eu35	Serratia liquifacca		Pr	11
E42	Ent. agglomeniani.se		Pr	13 .
E u45	Ent. cloacae
El54	Ent. agglo	-
Eu65	Ent. aerogenes	-
Eu66	Ent. agglo
Eu74	Ent.. hafnia
	-' ~..... ......

Serratia sp.

Klebsiella

Klebsi <] la axitob.is

Shigella bovde

E. coli

E. coli

Proteus vulgaris

Ent. cloacae

Serratia liquifacie is APO 0 0 0 9 8

Serratia sp.

Proteus mirabilis

Proteus mirabilis

TABLE 3

-38-

SALMONELLA SUPPLIED BY THE MEAT HYGIENE DIVISION'S SALMONELLA TYPING LABORATORY, THE DEPARTMENT OF VETERINARY SERVICES, MINISTRY CF AGRICULTURE ZIMBABWE ISOLATED FROM VARIOUS SOURCES. TESTED AGAINST THE

SALMONELLA-SPECIFIC DNA PROBE USING DNA-DNA COLONY HYBRIDIZATION.

SALMONELLA TYPE	RESULT
S typhi	+
S paratyphi B	+
S paratyphic	+
S infantis	+
S newington	+
S mobeni	+
S bron	+
S Montevideo	+
S banana	+ .
S mi n i	+
S bleadon	+
S cecro	+
S anatui.i	+
S difftor	+
S niqeria	+
S pretoria	+
S Zanzibar	+
S nideau	+

-39S raus +

S risscn +

S limbo +

S adeoyo +

S Cairo +

S menston +

S paratyphi A +

S kiel +

S colindalc +

S cquatonia +

S esscn +

S bulawayo +

S dares salam +

S Johannesburg +

AP 0 0 0 0 9 8

I

ANALYSIS BY X TEST OF COMPARISON OF THE RESULTS OBTAINED BY CLASSICAL MICROBIOLCGICAT METHOD AND BY THE USE OF SALMONELLA - SPECIFIC DNA PROBE IN DETECTING PRESENCE OF

SATMONELLAE spp IN VARIOUS SAMPLES

t: ~>»j nj ο Ό o •Η || rH 144 •Η Ο 4J •H 43 in (u	Very highly significant	Very highly significant	Very highly significant	.Very highly significant	Very highly significant	Very highly significant
Γ—1 03 CM	8*09	8*68	90.6	262.2	in VO m	in o VO Γ-
	Percent Positive	1.6	1.6	I 5.3	2.1	5.9	3.71
CLASSICAL MICROBIAL METHOD	Number Negative	147	i—1	! 189	489	376	1 248
Number Positive	m	m	r—I r—j	r—1 r—1	CM ·	CM m
Percent Positive	CO cn	o in	Γ	VO •tr	69.8	52.6
c o λ CJ co s CU	• Nuirber Negative	m σ\	in r-	105 .	270	121	<tr VO .. vo
Number ’ Positive	r- in	in	LD σ\	230	279	736
tn U-l <u 0 6 § ε cn	150	150	200	500	ί 400 | I	1 400
ω a & cn	δ 03	< MEAT and BONEMEAL	SWABS FRGM VARIOUS SITES and MATERIAIS	a el 05	DIARRHOEAL SAMPLES	1 Total number of . samples tested
sgonpoad grera pur grew	saeipo	soTduirg -[cotutid

-41REFERENCES CITED

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f iicroLiol. 1C: 333-342.

Cady, P., Dufour, 5.W., Show, J. and Kraeger, S.J., 1373. Electrical impendence measurements : rapid method for detecting and monitoring micro-organisms. J. Clin. Microbil. 7: 265-272.

Chen, C.H. and Thomas, J.C.A. 1330. Recovery of Di.’A segments from, agarose gels. Anal. Bi ocher.:, 101: 333-341 .

D'Aoust, J.Y. and I'.aishment, C., 1379. Pre-enrichment conditions for effective recovery of Salmonella in foods and feed ingredients. J. Focd-Prot. 42: 133-157.

Edel, I.', and Kampel-Macher, E.H. 13G3. Salmonel la European Laboratories using standard techniques. 4b 297-306.

Fitts, R., Diamond, M., Hamilton, C. and fieri hybridization assay for detection of Salmonellag Environ. Microbiol. 45: 1145.

isolation in nine

Bulletin of the '..HO , fi., * 1933. DNA-SfiA spp in foods. A.opl.

AP 0 0 0 0 9 8

Food and Drug Association, 1375. Bacteriological analytical manual for foods 4th ed. Association of Official Analytical Chemists. Washington

D.C.

Godson, G.fl. and Vapnek, D. 1573. A sit.,pie method for preparing large amounts of 174 RFI supercoiled DNA. Biochem. Biophys. Acta. 253:

516Hill, Fi.E. 1331. DNA hybridization method for detection enterotoxigenic Eschenichia coli in human isolates and its possible application to food samples. J. Food Safety; 3j 233-247.

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-42environmenta1 swab samples for Salmonellae. Appl. Microbiol. 26: 268-270.

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Mohr, H.K., Trenk, H.L. and Yeterian, H..1574. Comparison of fluorescent antibody method and enrichment serology for the detection of Salmonel la. Appl. Microbiol. 27: 324-32S.

Moseley, S.L., Hug, I., Alim, A.R.M.A., SO, [1. Samcdpour Motalebi, (i. and Flakow, S. 1580. Detection of Esclier iciiia coli by Di.’A colony' hybridization J. Infect. Dis. 142: 892-82-3.

Munson, T.E., Schrade, J.P., Disciello, N.E., Fantasia, L.D. Jr., Hartung, W.H., and O'Connor, J.J., 1276. Evaluation of an automated fluorescent antibody procedure for detection of Salmonella in foods and feeds.

Appl. Environ. Microbiol. 31: 514-521.

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Poeir.ia, P.L. and Si Hiker·, J.H., 1276 . Salmonel la. pp 301-323. In M.L. Speck-(ed.). Compendium of methods for the microbiological examination of foods. American Public Health Association Inc.

Washington D.C.

Silliker, J.H., Schmall, A. and Chill, J.Y. 1266. The fluorescent antibody technique as a means of detecting Salmonella in feeds. 2·

Food Science 31: 240-244.

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371-380.

Sperber, W.H., and Deibel, R.H., 1262. Accelerated procedure for Salmonella detection in dried foods and feeds involving only broth cultures and serological reactions. Appl. Microbiol. 17: 533-532.

(

-43Stev.-art, B.J., Eyles, 11.J. and Murrell, ’J.G., 1580. Rapid radior.ietric niethod for detection cf SaImonel la in foods. Appl. Environ.

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Thomason, 8., 1531. Current status of imrxinof luorescerit methodology for Salmonel la. J. Food Prot, 44: 381-334.

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27: £62-363.

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Claims (9)

1. A pure Salmonella DNA fragment comprising either:

a) the DNA sequence introduced into plasmid pNO 1523 carried by E. coli MC 1009 identified by accession ηο//_τ , SAL. 15DNA probe/88.

b) DNA sequences which will hybridize under low stringency conditions to the chromosomal DNA sequence of ail Salmonel]ae.

2. A DNA fragment according to Claim 1 which is derived from S. typhimurium.

3· A DNA fragment according to Claim 2 obtained by restriction of the S. typhimurium chromosome with Hpa I.

4· A DNA fragment according to any preceding claim which is about 2.3· Kb in length.

5· A DNA fragment according to any preceding claim which is labelled in order to facilitate detection.

6. A DNA fragment according to Claim 5 preferably labelled with non-isotopic materials such as biotin or sulfo-probe DNA systems.

7· A plasmid which contains an inserted DNA sequence in which the inserted DNA sequence is a DNA fragment according to any preceding claim.

8. A host cell transformed with a plasmid or DNA fragment according to any preceding claim.

AP000098 ./

BAD ORIGINAL M

-29. A host, cell according to Claim 8 which is a bacterial cell.

10. A host cell according to Claim 9 which is E. coli MC 1009·

A Salmonellae species - specific DNA fragment which is essentially pure and capable of hybridizing to the DNA of all Salmonellae species.