WO2005028668A1

WO2005028668A1 - General method for enrichment and detection of pathogen bacteria

Info

Publication number: WO2005028668A1
Application number: PCT/DK2004/000648
Authority: WO
Inventors: Torben Jensen
Original assignee: Bioline Aps
Priority date: 2003-09-25
Filing date: 2004-09-23
Publication date: 2005-03-31
Also published as: DK200301392A

Abstract

The invention concerns a horizontal fast-method for enrichment and detection of pathogen bacteria in a sample, including faeces.

Description

General method for enrichment and detection of pathogen bacteria.

Application of the invention

The invention relates to a horizontal method for enrichment and detection of pathogen bacteria including Salmonella, Listeria, E.Coli, Campylobacter, Yersinia etc. in a sample including, faeces, food, feed, swabs, HACCP (Hazard Analysis Critical Control Point), pharmaceuticals, food additives, additives and other products which can be consumed by or get in contact with humans, domestic animals or pets including feather, dust, down or seawage.

Technical stage

The presence of pathogen bacteria e.g. salmonella in food and feed for domestic animals constitute a problem for public health. Salmonella belongs to the group of gram-negative enterobacter and their presence in the intestine often leads to gastrointestinal difficulties for humans and animals. The symptoms are gastroenteritis sometimes as mild diarrhea but often also involving severe diseases. Salmonella and Campylobacter are the most common causes of diarrhea caused by bacteria. These bacteria infects humans and animals through the per oral route by consumption of contaminated food.

Many manufacturers of food and feed are obliged to deliver products free of pathogens and therefore analyse their production for the presence of pathogen bacteria.

All general methods for detection of pathogen bacteria in food, feed, faeces etc. are specified in the so-called horizontal methods - that are methods, which for each pathogen can be used for detection of the organism in a broad range of different items of food, feed, sewage etc. Previously the so-called vertical methods were often applied for detection in only a narrow range of items as e.g. fresh meat.

Many horizontal methods have eventually been converted to standardized ISO-methods, which are the methods laboratories needs to use in the particular countries. The ISO- methods are also called reference methods but there exists other corresponding methods of reference e.g. in the USA: BAM (Bacteriological Analytical Manual), in Scandinavia: NMKL (Nordisk Metodik Komite for Levnedsmidler), in Germany (LMBG §35). Further other national sub variants excists. All reference methods are based on well-known and relatively safe cultivation- and enrichment techniques but they all suffer from the disadvantage of being very labor intensive and slow. The classical method of analysis of salmonella according to the NMKL 71-1999/ISO 6579:2002 is based on a 16-20 hours pre-enrichment in a non-selective growth medium to start growth of the bacteria. The following day a small volumen from the grown pre- enrichmentbuffer is transferred to one or several selective enrichment media allowing growth of salmonella but no or only limited growth of other bacteria. After additional incubation 1-10 ul is seeded on at least 2 indicative agars, which is then incubated for 24 hours before reading. The result of the analysis of the sample is in this way indicatively obtained after ca.3 days. If the indicative agars contain suspicious bacterial colonies at least 1 day additional work is required before a definitive positive result can be claimed.

The classical methods of reference, which can be used generally (horizontal) for detection of pathogen bacteria, are restrictive regarding permitted deviations from determined parameters including temperatures, periods and transfer volumes.

The methods of reference do not provide the user any options to change these defined parameters. Therefore periods, volumes or temperatures can not be changed. Thus an expert in the field cannot change these parameters without major investigations and following national/international approvals, which is not a common procedure.

The classical methods of analysis have the disadvantage of being very slow but also the disadvantage of being very labor-intensive. Therefore many experts and testkit providers have attempted to shorten the time of detection maximally without loosing reliability in the methods. But reducing the time of detection has only partly been achieved and only in and only for samples which have limited amount of non-target organisms present.

For many methods a major amount of non-target bacteria will have the unfortunate characteristic to shield the signal from the pathogen target-bacteria. This has the unfortunate consequence, that a laboratory of analysis examining such a sample, which is not solely infected by pathogen target-bacteria but also a larger amount of non-target bacteria, may erroneous characterize the sample as free of pathogen.

The need of a fast and reliable detection of the presence of pathogen bacteria in food, feed, domestic animals, faeces, seawage etc. is of great economical importance within many producers including slaughterhouses, manufacturers of eggs, manufacturers of food in general and feed producers.

The duration of obtaining a result can be divided in a period of enrichment to multiply the target-organism and a following peroid of detection. A shortening of the time of obtaining the result can be achieved by shorten the period of enrichment and/or the time of detection, where the period of enrichment can be subdivided in a period of pre-enrichment and a period of selective enrichment. The period of selective enrichment can be divided into two or consecutive enrichments.

Several investigators have attempted to reduce the time of pre-enrichment

D'Aoust and Maishment ,1979 investigated the reduction of the pre-enrichment period to 6 hours in different non-selective media. They found, that it is not possible to shorten the duration of pre-enrichment to for example 6 hours since a shortening will lead to an unacceptable low rate of recovery of the target-bacteria.

Chen, 1993 investigated pre-enrichment in selective media and concluded that after a shortening of the duration to 6 hours it was not possible to obtain positive samples.

Patil, 1986 found in a similar way that a shortening of the duration of pre-enrichment in principle is not possible.

Attempts to reduce the time of the selective enrichment were neither a practicable and safe solution.

Lubenow, 2002 showed that a pre-enrichment of normal duration followed by a shortened enrichment similar to a selective enrichment step did no produce a sensitivity as good as the method of reference.

Thus it appear that it is not possible to reduce the duration of enrichment only by changing the duration of pre-enrichment or the duration of selective enrichment.

Attempts to force the enrichment by increasing the transfer volume from the pre-enrichment to the selective enrichment have shown that a change of the transfer volume from the usual 100 uL preenriched sample into 10 mL RVS produced worse results for salmonella. The experiment was carried out both with smaller and larger transfer volumes. (Fricker, 1985). Thus it appear that it is not possible to reduce the time of enrichment only by changing the size of the transfer volume nor by increasing the transfer volume from the pre-enrichment to the container of the selective enrichment.

Attempts to predict the optimization of the enrichment by devising mathematical models of the number of bacteria has lead to no conclusion.

D'Aoust, 1991 concludes in a review article on the survival capacity / adaptability of the salmonella bacteria that: "Mathematical models to calculate the enrichment of salmonella bacteria can not be applied for prediction of the actual number salmonella bacteria in cooled items."

This is supported by Chung K. C and Geopfert, 1970, who claimed that: "it is impossible to predict or extrapolate results from world of laboratory to real samples". Thus these experiments shows that one cannot state the number of bacteria in the real world by applying laboratory- or mathematical models.

In 1987 Van Schothurst suggests a development of the classical salmonella methods. But in spite of a massive product development it has still not been possible to provide a general and/or horizontal method, which can clarify the presence of a single pathogen bacterium in a product of investigation near to 24-30 hours or less, when the method must have a sensitivity which is only approximately equal to acknowledged methods of reference.

From the literature it cannot be deduced which preconditions, which must be fulfilled to provide a horizontal procedure for enrichment of pathogen bacteria including Salmonella, Listeria, E.coli, Campylobacter, Yersinia etc. When the procedure must be employed for a safe detection of a single bacterium in a sample of 25 gram after a single day and night, when the sample can be faeces, food, animal feed and other products, which humans or domestic animals and pets can consume or get in contact with. Since the laboratories do not know if the sample to be analyzed contains or has been exposed to a possible faecal contamination it is indispensable claim for the procedure that a selective enrichment is carried out to consider the growth of target-bacteria simultaneous to the growth repression of non-target bacteria.

To reduce the total time of analysis one has besides the theoretical possibility to reduce the duration of enrichment also a theoretical possibility to reduce the duration of detection. The latter can be achieved by developing more and more sensitive methods of analysis, which can demonstrate the presence of fewer and fewer target bacteria in a sample. Very sensitive equipment of analysis has besides being very expensive to purchase and employ, often the disadvantages that the background noise also can be enhanced when the target- signal is enhanced.

By employing specific antibodies it has become possible to shorten the duration of analysis to about 2 days and nights after classical- or classical-like enrichments. The most used technology has hitherto been ELISA (Enzyme Linked Immunosorbent Assay) and attempts to further shortening enrichment duration followed by specific ELISA, produced results with varying agreement. Pelton 1994, Peplow 1999, Poppe 1996. Also DNA/RNA hybridization has been employed likewise with a duration of analysis of ca. 2 days and nights. The same applies to methods employing measurement of conductance/conductivity.

The latest developments have been based on PCR technology (Polymerase Chain Reaction), which after a single non-selective pre-enrichment can detect the presence of a single pathogen bacterium in an investigated product in ca. 20-24 hours. However this technology require special organization of the laboratory and expensive instrumentation. This has contributed to the very limited prevalence of the PCR technology. The enrichment applied for PCR technology furthermore implies that the sample contains a limited amount of other bacteria, not disturbing the result of analysis.

If the sample contains only few target-bacteria, it can be difficult to detect the these if the sample also contains a large number of non-target bacteria as these may interfere with the signal from the pathogen target-bacteria.

If the ratio between target-bacteria and non-target bacteria exceeds 1:1000 a false result may be the consequence. This devalued ratio is obtained for the cultural methods using indicative agars, but also ELISA and PCR can be affected.

Sibley, 2003 found that PCR cannot be applied for faecal analysis if not a step of selective enrichment was inserted to connect the pre-enrichment and the step of detection. In a similar way Ziemer, 2001 concluded to obtain correct results the cultural methods couldn't be shortened.

Thus samples contaminated with non-target organisms in high number, has therefore the very unfortunate consequence that the result of analysis will characterize the samples as pathogen-free (= false negatives), since the signals from the pathogen target-bacteria are not detected caused to the disturbing effect from the other non-target bacteria during the enrichment. This misjudgment of results can in the worst scenario lead to economical consequences. The selective step is critical because it shall repress the non-target bacteria. (Michael, 1999).

Until the appearance of enrichment according to the present invention, comprehending a selective enrichment of the target-bacteria, it has not been possible to analyze strongly contaminated samples with an acceptable recovery in less than 30 hours.

Certain types of samples contains no or very small number of non-target bacteria, while other types of samples may contain larger numbers of such bacteria. Usually it is not very difficult to find the pathogen bacteria in the sample material with no or very small number of non-target bacteria. However the interference of analysis will rise with the number of non- target bacteria. Certain types of samples for example samples obtained from faeces or slaughterhouses may contain larger numbers of non-target bacteria, which in most systems of analysis may cause interference making the result less reliable. Occasionally samples of meat also may contain significant numbers of no-target bacteria, which originate from the removed entrails etc.

The general description of the invention In connection with the development of selective nutrient media / growth media for pathogen bacteria including salmonella bacteria, the inventor has on several occasions titrated nutrient media / growth media for the number of pathogen bacteria. In the light of this, the inventor has succeeded in exposing the absolute number of target-bacteria at any time in actual nutrient media / growth media in presence of different sample matrices including food, feed, offal, faeces etc. and with various level of other bacteria of relevance in both the pre- enrichment media and the selective enrichment media.

The inventor has surprisingly demonstrated that even very low concentrations of pathogen bacteria has a surprisingly ability to quickly restitute for exponential growth, even when the samples are loaded with a large number of non-target bacteria, implying that the target- bacteria are offered optimal conditions of growth, including elevated temperature over room temperature especially at the start of analysis. Furthermore the inventor has surprisingly demonstrated that the following have an unambiguous positive effect on the percentage of recovery: An unusual early transfer of a partial volume (transfer volume) from the pre- enrichment to the selective enrichment, a transfer of an unusual large amount of transfer volume and especially for large transfer volumes, that the receiving buffer has a concentration accounting for the dilution the transfer volume entails.

By combining all 3(4) essential factors contemporary, the inventor has surprisingly succeeded to procure a very fast and safe enrichment of pathogen bacteria according to the invention. Namely:

• Optimal (high) initial temperature

• Large transfer volume

• Early time for transfer (•) A selective media accounting for the large dilution caused by the transfer volume.

This new general procedure for enrichment of pathogen bacteria according to the invention, is significantlt faster than the general standard methods the laboratories of analysis are obliged to apply for enrichment of pathogen bacteria from a sample.

The inventor succeeded to reduce the general time of enrichment of pathogen bacteria from ca. 44 hours to as low as ca.24 timer.

An evidence for the quality of the faster enrichment method, SELECTA, according to the invention, was carried out by ASEPT, L'Hygiene dans la Qualite, 53020 Laval Cedex,

France, which is an impartial laboratory. ASEPT has performed a comparison of methods between the Bioline SALMONELLA OPTIMA and the Bioline SALMONELLA SELECTA, where OPTIMA is based on a classical enrichment both on temperature, times and volumes.

A test of 374 samples yielded 6 false negative results with OPTIMA, the classical enrichment combined with the Bioline ELISA, while the same samples yielded no false negative, when the SELECTA method according to the invention with the short enrichment of 24 hours was applied.

It was possible to demonstrate that the method is unique since the same sample and the same pre-enrichmentbuffer was applied for both systems, i.e. the starting material was the same. The only difference was the size of the transfer volume, the time of transfer and the following selective buffer.

This new general procedure for fast enrichment of pathogen bacteria has the immediate great advantage that the enrichment can be shortened with at least one workday and that ELISA methods can be used for detection of e.g. Salmonella bacteria in 20-28 hours maybe even shorter if more sensitive detection methods are applied.

Hereby the ELISA-methods becomes a real alternative to the very expensive and in certain cases uncertain PCR-methods, when a clarification of a possible content of pathogen bacteria in a product is required within 20-28 hours.

Following the actual procedure of enrichment of Salmonella obtained certification and been recognized for detection of Salmonella in all types of food and feed - thus a horizontal methods, there exist no other alternative to this invention, when a result is required within 20 to 28 hours.

The nearest alternative for a horizontal method is the conventional methods of reference, which on the earliest provide a result after 60 hours as the most recent regulations command.

It will be obvious for an expert in the field that this general procedure for enrichment of pathogen bacteria, where the result of the enrichment are to be applied for the clarification of the presence of pathogen bacteria in a product, also can be used as preparation of sample for any method of analysis, including ELISA-methods with very sensitive markers e.g. fluorescence, PCR-analysis with more - hereby it will be possible to shorten the total time of analysis from sample preparation to the final result to one day and night.

It has surprisingly appeared that the enrichment procedure for pathogen bacteria according to the invention not only has a sensitivity, which is fully equal to the conventional methods. The enrichment procedure of pathogen bacteria according to the invention has in repeatedly studies appeared to be significantly better than the procedures applying 40-48 hours of enrichment, especially if high numbers of non-target bacteria are present.

The general method for enrichment of pathogen bacteria according to the invention has been validated by AFNOR, NordVal and approved in line with other methods for enrichment of salmonella bacteria.

A successful detection of a positive sample requires that at least 1 target-bacteria is present in the transfer volume taken from the preenrichment buffer. Therefore it is essential to ensure initial and optimal growth conditions for the target bacteria in the pre-enrichment buffer. Many pathogen bacteria grow fast at about 37°C other bacteria grow better at 41 °C. If the initial temperature of the preenrichment buffer is room temperature the multiplication rate will be slower compared to the best possible temperature. The corresponding values for the duration of pre-enrichment and the size of the transfer volume shall ideally be adjusted in a way that the transfer volume would include at least 1 target-bacteria to ensure the inoculation of the selective medium and that the final concentration of the pathogen target- bacteria exceeds the detection limit of the method of analysis, in those cases when the sample is positive. The detection limit for most applied methods is from 10⁵/mL-10⁶/mL, (Huang, 1999) PCR has a lower sensitivity of 500-1000 bacteria/mL

To consider optimal growth conditions for the pathogen target-bacteria the receiving selective enrichment buffer is ideally to be adjusted to the dilution the transfer volume will cause. If a transfer volume of 20 mL is chosen to be added to 200ml of selective media, there must initially be media components corresponding to 220 mL in the selective enrichment container.

The actual procedure has been validated after NordVal protocol using the Bioline Salmonella ELISA kit for detection. The method of reference was ISO 6579:2002. From 179 positive samples of which 30% were naturally contaminated and the rest contaminated with 1-10 cfu per sample of various salmonella strains, the procedure according to the invention obtained 100 % accordance with the method of reference. It was the same case for the 187 negative samples, which all were in accordance by both the actual procedure and the method of reference.

EXAMPLES OF PERFORMANCE

EXAMPLE 1

Determination of time of initial incubation necessary for positiv identification of

Salmonella. 25 grams of raw milk was added 225 ml of Buffered Peptone Water pre-warmed to 37°C and added 2.5 cfu Salmonella Typhimurium.

In parallel 25 grams of chopped raw meat was added 225 ml Buffered Peptone Water pre- warmed to 37°C and added 2.5 cfu Salmonella Typhimurium. In parallel 25 grams of chicken meat was added 225 ml Buffered Peptone Water pre- warmed to 37°C and added 2.5 cfu Salmonella Typhimurium. The samples were stomached and placed in an incubator at 37°C. A volume of 20 mL was withdrawn at the time intervals 1 , 2, 3, 4, 5 and 6 hours and immediately transferred to single portions of 200 mL Rappaport Vassiliadis 10 % more concentrated and pre-warmed to 41.5°C prior to the addition of the transferred volume. The second enrichment was incubated for 19 hours in an incubator at 41.5°C. Ten ul of the samples were withdrawn for detection by culture and 1 mL was then withdrawn for boiling prior to the ELISA test.

OD450nm values higher or equal to 0.200 are considered positive. It is shown that a positive identification can be achieved for chopped raw meat after only 2 hours of pre-incubation and for chicken and raw milk after only 3 hours. Assuming that the final volume is 250 mL and that at least 1 cfu was contained in the transferred volume of 20 mL, 12.5 multiplications took place after 2 and 3 hours, respectively, according to the type of sample which indicates an expected time of doubling of ca.30 min. in average, which is in agreement with the reports in the literature. We conclude that a pre-enrichment of 6 hours contains a safe margin for withdrawal of this large transfer volume.

EXAMPLE 2

Determination of the volume of transfer necessary for positive identification of

Salmonella. 25 grams of raw milk was added to 225 mL Buffered Peptone Water pre-warmed to 37°C and added 3.6 cfu of Salmonella Anatum. In parallel 25 grams of chopped raw meat was added to 225 mL Buffered Peptone Water pre-warmed to 37°C and added 3.6 cfu Salmonella Anatum.

In parallel 25 grams of chicken meat was added to 225 mL Buffered Peptone Water pre- warmed to 37°C and added 3.6 cfu Salmonella Anatum.

The samples were stomached and placed in an incubator at 37°C. A volume of 0.1 to 20 mL was withdrawn after 4 hours and immediately transferred to different portions of Rappaport Vassiliadis. The second enrichment was incubated for 19 hours in an incubator at 41.5°C and 1 mL was then withdrawn for boiling prior to the ELISA test. Similarly there were withdrawals for detection by indicative agars.

OD450nm values higher than or equal to 0.200 are considered positive. * Selective enrichment media was adjusted to 10 % higher concentration. It is shown that a positive identification can be achieved with a transfer volume of 8 mL and 20 mL for chicken, chopped beef meat and raw milk even after only 4 hours of pre- incubation. Also we conclude that 20 mL from the pre-enrichment is a safe margin for withdrawal of volume from the sample.

Chicken, raw milk and chopped beef Experimental investigations shows as expected that the rate of recovery in chicken, raw milk and chopped beef is depending on when the transfer volume is withdrawn from the preenrichment buffer and how large a transfer volume there is withdrawn from the preenrichment buffer and transferred to the selective growth medium. But after a contamination with only 2.5 cfu (colony forming units) it has surprisingly shown possible to detect positive samples with 100 % certainty after only 3 hours of pre-incubation for all investigated samples. For chopped beef meat is it possible to detect positive samples with 100 % certainty after only 2 hours of pre-enrichment, if the sample of 25 grams is applied to 225 mL preenrichment bufferent buffer at a temperature of 37°C and when a transfer volume of 20 mL is transferred from the pre-enrichment buffer to a selective enrichment buffer of 200 mL at 41.5°C for ca. 19 hours.

Furthermore experimental investigations show that the rate of recovery is 100 % for salmonella in chicken, raw milk and chopped beef meat when contaminated with only 3.6 cfu (colony forming units) in 25 grams test material in 225 mL pre-enrichment buffer at 37°C after only 4 hours, when a transfer volume of 8 mL is withdrawn from the pre-enrichment buffer and when the selective enrichment buffer is of 200 mL at 41 ,5°C. For chicken and chopped beef it is possible to detect positive samples with 100 % certainty by withdrawing 0.5 L and 1.0 mL transfer volume, respectively, in 50 L and 100 mL selective enrichment buffer, respectively, at 41 ,5°C for 19 timer.

It will be obvious for an expert in the field that a nutrient substrate, which offers target- bacteria considerable better growth conditions, with great advantage can dictate a smaller transfer volume, and earlier time for transfer of the transfer volume from the pre-enrichment buffer, if it is guaranteed that the transfer volume contains at least one pathogen target- bacteria.

Investigation of faeces.

Investigations of faeces from domestic animals has gained larger importance since by testing the occurrence of e.g. salmonella in faeces from livestocks, a logistically control is possible of the appearance of the livestocks to the slaughterhouse. In this context it is possible to work with "clean" lines and "non-clean" lines in other periods with following cleanup of the slaughtering line. However, there is an analytical problem in the detection of Salmonella in animal faeces since this type of sample in particular is extremely loaded with non-target bacteria, which disturbs successful detection.

Numerous studies and developments of methods have been carried out exactly with this type of samples and many countries have now agreed to apply the MSRV method. This method is about to be included in the existing ISO 6579:2002 standard for detection of salmonella in food and animal feed. So there exist a broad agreement that the MSRV method is superior to other methods of reference for faecal samples.

The actual procedure for the enrichment of pathogen bacteria according to the invention, where the verification of salmonella applied the ELISA-kit from Bioline ApS, was applied in a performance test organized by the NRL-Salmonella, the Danish Veterinary Institute (Report 3. Ringtrial for evaluation of bacteriological method for isolation of Salmonella bacteria from domestic animals by Dorte Lau Baggesen and Gitte Sørensen).

Under these very difficult circumstances the actual procedure according to the invention, which for verification of salmonella applied the ELISA-kit from Bioline ApS, demonstrated to be superior to all other comparable procedures since all 18 positive samples were detected with 100 % unambiguous values of results. The average rate of recovery by the MSRV methods in de other 13 laboratories were averaged to only 81 % varying from 39 to 94 % as the best. Furthermore two commercial PCR methods were applied in two of the participating laboratories. One concluded no positives and the other laboratory found 6 positive.

Below is shown results for the MSRV and the Bioline SELECTA methods. The samples were coded and unknown until ca. 1 month after submitting the results.

The rapid methods attempted to be developed in the recent years suffers from difficulties due to high levels of non-target bacteria, since to obtain shorter periods of enrichment less selective media are applied or application of selective media are totally omitted, which will speed up the growth of non-target organisms.

The preferred type form of execution

By applying the horizontal procedure for enrichment of pathogen bacteria e.g. salmonella bacteria according to the invention, you can with unfailing certainty achieve a correct interpretation concerning the presence or absence of a single salmonella bacterium in a sample, within the area of application of the invention, by applying 25 grams of test material to 225 mL pre-enrichment buffer pre-warmed to 37°C, withdraw a transfer volume of 20 mL from the pre-enrichment buffer after 6 hours and transfer this volume to 200 mL "110 % concentrated" pre-warmed SELECTA selective growth medium with following incubation at 41,5°C for 18 hours, when the SELECTA salmonella ELISA test kit from Bioline ApS is applied for detection.

The specific description of the invention

The purpose of the invention is to provide a horizontal procedure for determination of the presence of pathogen bacteria including Salmonella, Listeria, E.coli, Campylobacter, Yersinia along with others in a sample, which can be faeces, food, feed and other products which can be consumed by or get in contact with humans, domestic animals or pets, where other products include but is not restricted to feather, dust, down, sewage and HACCP- samples, which procedure comprehend:

- an enrichment of the pathogen target-bacteria, whenpresent in the investigated sample in a pre-enrichment buffer,

- a transfer of a transfer volume (a partial volume of the pre-enrichment buffer) to a selective enrichment buffer(s), which selective enrichment buffer(s) as a minimum contains an agent, which is not tolerated and/or is less tolerated by non-target bacteria (other bacteria) and/or other selective pressure on the non-target bacteria e.g. pH, osmosis,

- if necessary followed additionally by one or more selective enrichment buffers

According to the invention this is achieved by a contemporary combination of 3(4) essential parameters for an optimal growth of the investigated target-bacteria, which comprehend:

- that the pre-enrichment medium is pre-warmed to above room temperature, preferably above 28°C,

- that the transfer volume from the pre-enrichment to the selective enrichment is carried out after less than 14 hours and

- that the transfer volume is preferably at least 1 mL resp. at least 1/200 of the selective growth medium.

And especially for large transfer volumes,

- that the concentration of the growth medium in the receiving selective enrichment buffer has a concentration of the growth medium which is equal to (or considerable higher than) recommended by the manufacturer of the growth medium and/or approved procedures for the pathogen target-bacterium after the transfer volume has been added to the selective growth medium.

The pre-enrichment medium can with advantage be pre-warmed to a temperature preferably above 30°C, more preferably above 34°C, respectively to 37°C +/- 3°C, preferably 37°C to stimulate the initial growth of the target-organism.

It greatly increases an early recovery considerably, that the transfer of the transfer volume from the pre-enrichment to the selective enrichment preferably is performed after less than 12 hours, more preferably after 6 +/- 4, more preferably after 6 +/- 2 timer, most preferably after 6 hours. With particular samples it increases an early recovery considerably, that the transfer of the transfer volume from the pre-enrichment to the selective enrichment preferably is performed after less than 2 hours.

It increases an early recovery percent considerably, that the transfer volume from the preenrichment to the selective enrichment preferably is of at least 2 mL, preferably is of at least 5 mL, preferably, is of at least 10 mL, preferably is of at least 20 mL, preferably is of at least 50 mL, resp. preferably at least 2/200, preferably at least 5/200, preferably at least 10/200, preferably at least 20/200, preferably at least 50/200 of the selective growth medium.

The procedure according to the invention is so sensitive that it can be applied for determination of the presence of a single pathogen bacterium in an investigated product.

The procedure according to the invention is applicable for detection of all pathogen bacteria including Salmonella.

The procedure according to the invention is optimized by application of SELECTA BIOLINE substrate for the selective enrichment.

Furthermore the procedure can according to the invention with advantage apply 2 or several consecutive and/or parallel selective growth media. The application of 2 or several consecutive selective growth media can ensure a constant selective pressure for a longer period, since the selective pressure often decreases in time with that the selective agent(s), which exactly should impede the non-target bacteria from growing, gets neutralized e.g. by adsorption. Therefore the growth of the target-bacteria may be improved compared with the growth of the non-target bacteria by applying successive selective growth media. The application of 2 or more parallel selective growth media can by be applied when detection of 2 or more different target organisms in the same sample is required.

The initial enrichment medium can by advantage be added growth enhancers, resuscitation enhancers and/or selective and/or partly selective compounds such as selenite, tetrathionat, Novobiocin, antibiotics, Brilliant Green and/or Malachite Green to ensure a high percent of recovery. It increases the percent of recovery considerable to shorten the total selective enrichment time to 1 , 2, 4, 6, 8 or 17 hours.

The selective growth medium or media can by advantage contain one and/or more selectiv(e) agent(s) including but not restricted to: tetrathionat, brilliant green and/or malachite green.

After the completion of the enrichment a method of analysis can be applied for the detection step including but not restricted to: affinity binding technique such as enzyme immunoassays (ELISA) based on antigen- antibody reactions, antigen-antibody reactions which involves fluorescence, luminescence, evanescent waves, plasmon resonance, latex agglutination, electrochemical immune detection, techniques with immunomagnetic capturing, techniques with lateral flow, DNA hybridization based on specific sequences of salmonella DNA molecule, RNA-DNA, RNA- RNA, Polymerase Chain Reaction (PCR) based on multiplication by specific DNA primers, conductance measuring methods based on change in the electric resistance in special growth media, microscoping, techniques with micro arrays, CCD camera technique, enzyme immuno technique based on chromogene, fluorescence, luminescence, radioactive signal generating response, semisolid agars and solid agars or combination with further enrichment procedures.

The procedure for enrichment and detection of pathogen bacteria in a sample according to the invention is optimized by application of ELISA test kit from Bioline ApS.

It increases an early percent of recovery considerable when the procedure according to the invention is carried out automatically or semi-automatically i.e. independent or by limited application of manual workload by e.g. transfer of the transfer volume from the preenrichment to the selective enrichment and/or a volume from the selective enrichment container to inactivating/lysation/boiling/radiation/killing and/or of the sample of analysis from destruction to the analytic detection step. Definitions:

The validation requirements of NordVal for approval of a microbiological method as an acceptable method imply: that, the relative sensitivity (percent of recovery) is at least 95 %, i.e.: that the maximal occurrence of false-negative samples (i.e. samples which contains pathogen bacteria is analyzed as pathogen-free) is 5 % and that the maximal occurrence of false-positive samples (i.e. samples which do not contain pathogen bacteria is analyzed as infected by pathogen).

List of references

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D'Aoust J-Y, 1991 , International Journal of Food Microbiology 13 pp. 2007-216 Psychrotrophy and Foodborne salmonella.

Chen H, Fraser A D E, Yamazaki H, 1993 International Journal of Food Microbiology 18 pp. 151-159. Evaluation of the toxicity of Salmonella for the shortening of enrichment period.

Chung K C, Geopfert J M, 1970 Journal of Food Science 35 pp. 326-328: Growth of salmonella at low pH.

Fricker C R, Quail E, McGibbon L, Girdwood R W A, 1985, J.Hyg. Camb.95 pp. 337-344: An evaluation of commercially available dehydrated Rappaport-Vassiliadis medium for the isolation of salmonella from poultry.

Huang H, Garcia M M, Brooks B W, Nielsen K, Sze-Park N, 1999, International Journal of Food Microbiology 51 pp. 85-94: Evaluation of Culture enrichment procedures for use with Salmonella detection immunoassay.

Lubenow E, 2002 PhD arbejde, Tierartzliche Hochschule Hannover: Untersuchungen zum Nachweis von Salmonellen in natϋrlich kϋnstlich kontaminierten Lebensmitteln mittels kultureller Methode, ELISA, PCR, und Microarray.

Michael G B, Cardoso M, Costa M, 1999, Proceedings of the 3'rd International Symposium on the Epideomiologi and Control of Salmonella in Pork, Washington D.C. August 5-7, 1999: Comparison of Selenite Cystine Broth, Tetrathionate Broth and Rappaport-Vassiliadis Broth for the recovery of salmonella from Swine Feces.

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Pelton J A, Dilling G W, Smith B P, Jang S, 1994, J. Vet Diagn Invest 6 pp. 501-502 Comparison of a commercial antigen-capture ELISA with enrichmnet culture for detection of Salmonella from fecal samples.

Peplow M O, Correa-Prisant M, Stebbins M E, Jones F, Davies P, 1999, Applied and Environmental Microbiology 65(3) pp. 1065-1060, Sensitivity, Specificity and Predictive values of three Salmonella Rapid Detection Kits using fresh and frozen poultry environmental samples versus those of standard plating.

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Claims

CLAIMS OF PATENT

1. Horizontal procedure to enrichment and detection of pathogen bacteria in a sample including Salmonella, Listeria, E.coli, Campylobacter, Yersinia etc. from faeces, food, animal feed and other products which can be consumed by or get in contact with humans, domestic animals or pets, where other products include but is not restricted to feather, dust, down, seawage and HACCP-samples.

which procedure as a minimum include: - an enrichment of pathogen target-bacteria in a pre-enrichment buffer and

- a transfer of a transfer volume (a partial volume of the pre-enrichment buffer) to a for the target-bacteria selective enrichment buffer, which selective enrichment buffer as a minimum contains an agent which is not tolerated or is tolerated poorly by non-target bacteria (other bacteria) or other selective pressure on non-target bacteria e.g. pH and osmosis.

RECOGNIZED BY

- that the pre-enrichment medium is pre-warmed to above room temperature, preferably above 28°C, - that the transfer volume from the pre-enrichment to the selective enrichment is carried out after less than 14 hours and

2. Procedure according to claim 1 RECOGNIZED BY that the concentration of the growth medium in the receiving selective enrichment buffer has a growth medium concentration which is equal to or considerable higher than recommended by the manufacturer of growth medium or approved procedures for the pathogen target-bacterium after addition of the selective growth medium.

3. Procedure according to any of the previous claims RECOGNIZED BY that the preenrichment medium is pre-warmed to a temperature preferably above 30°C, more preferably above 32°C, most preferably above 34°C.

4. Procedure according to claim 1 RECOGNIZED BY that the pre-enrichment medium is pre- warmed to 37°C +/- 3°C, preferably to 37°C.

5. Procedure according to any of the previous claims RECOGNIZED BY that the transfer of a transfer volume from the pre-enrichment to the selective enrichment preferably is carried out after less than 12 hours, more preferably after 6 +/- 4 hours, more preferably after 6 +/- 2 hours, most preferably after 6 hours.

6. Procedure according to any of the previous claims RECOGNIZED BY that the transfer of a transfer volume from pre-enrichment to the selective enrichment preferably is carried after less than 2 hours.

7. Procedure according to any of the previous claims RECOGNIZED BY that the transfer volume from the pre-enrichment to the selective enrichment is preferably of at least 2 mL, preferably of at lest 5 mL, preferably of at least 10 mL, preferably of at least 20 mL, preferably of at least 50 mL, resp. preferably at least 2/200, preferably of at least 5/200, preferably at least 10/200, preferably of at least 20/200, preferably of at least 50/200 of the selective growth medium.

8. Procedure according to any of the previous claims RECOGNIZED BY that the procedure is applied for the determination of the presence of a single pathogen bacterium in an investigated product.

9. Procedure according to any of the previous claims RECOGNIZED BY that the pathogen bacterium is salmonella.

10. Procedure according to any of the previous claims RECOGNIZED BY that the SELECTA BIOLINE substrate is applied for the selective enrichment.

11. Procedure to enrichment and detection of pathogen bacteria in a sample according to any of the previous claims RECOGNIZED BY that a pre-enrichment buffer and at least 2 selective growth media are applied.

12. Procedure according to any of the previous claims RECOGNIZED BY that the initial enrichment medium is added growth enhancers, resuscitation enhancers or selective or partly selective compounds such as selenite, tetrathionat, Novobiocin, antibiotics, Brilliant Green or Malachite green.

13. Procedure according to any of the previous claims RECOGNIZED BY that the selective enrichment is shortened to 1, 2, 4, 6, 8 or 17 hours.

14. Procedure according to any of the previous claims RECOGNIZED BY that the second buffer applied for the enrichment of target-organisms contains tetrathionat, Brilliant Green or Malachite Green.

15. Procedure for enrichment and detection of (if any) pathogen bacteria in a sample according to any of the previous claims RECOGNIZED BY that a method of analysis is applied for the detection step including but not restricted to: affinity binding technique such as enzyme immunoassays (ELISA) based on antigen- antibody reactions, antigen-antibody reactions which involves fluorescence, luminescence, evanescent waves, plasmon resonance, latex agglutination, electrochemical immune detection, techniques with immunomagnetic capturing, techniques with lateral flow, DNA hybridization based on specific sequences of DNA molecule, RNA-DNA, RNA-RNA, Polymerase Chain Reaction (PCR) based on multiplication by specific DNA primers, conductance measuring methods based on change in the electric resistance in special growth media, microscoping, techniques with micro arrays, CCD camera technique, enzyme immuno technique based on chromogene, fluorescence, luminescence, radioactive signal generating responds, semisolid agars and solid agars or combination with further enrichment procedures.

16. Procedure for enrichment and detection of (if any) pathogen bacteria in a sample according to any of the previous claims RECOGNIZED BY that the ELISA test kit of Bioline ApS is applied for the detection step.

17. Procedure according to any of the previous claims RECOGNIZED BY that the transfer of the transfer volume from the pre-enrichment to the selective enrichment is carried out automatically or semi-automatically.