Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________

Specific and Sensitive Multiplex PCR Method for Detecting Salmonellae and Shigellae in Mayonnaise E. Villalobo Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Av Reina Mercedes, 6. 41012 Sevilla. Spain Bacteria from Genera Salmonella and Shigella can be naturally found in environment, animals, and food. These bacteria are pathogens to human, whose anomalous activity is responsible for major outbreaks. In the last half-part of the last century, food-associated infections caused by salmonellae and shigellae have increased. Along with this increment, the presence of these pathogens has been controlled systematically in many food types, such as mayonnaise. The widespread and validated method for detecting these pathogens in mayonnaise is microbiological culture however, DNA-based methods, such as polymerase chain reaction (PCR), have become very popular for microbial detection. We have developed a single-tube multiplex PCR method to detect salmonellae and shigellae. The method is applicable for the highly specific and sensitive detection of these pathogens with detection levels of 10-25 femtograms of isolated DNA or 2000 cells of S. typhimurium and S. dysenteriae per ml of diluting solution. The method has been successfully applied to detect these pathogens in diluted mayonnaise samples. It is concluded that the multiplex PCR is a method with enough specificity and sensitivity to be used for detecting the presence of salmonellae and shigellae in mayonnaise, and that it can save time and money compared to a single PCR method. Keywords IS200; Mayonnaise; PCR; Salmonella; Shigella; virA

1. Introduction Mayonnaise is a cold sauce whose main ingredients are egg, vegetal oil, vinegar or lemon juice, salt, and sometimes herbs or spices. This is a homemade food in Spain, Italy, and France however, the industrial manufacture accounts for the bulk worldwide consumption. Mayonnaise is an edible and stable emulsion where the fat, provided by the vegetal oil, is the discontinuous phase; the water, provided by the egg, is the continuous phase; and the lecithin, also provided by the egg, is the emulsifier. The manufactured mayonnaise is a rich food that sustains microbial growth. Its initial microbial flora [1] depends upon raw ingredients and is mainly composed of yeasts (Saccharomyces), lactic bacteria (Lactobacillus), and spore-forming bacteria (Bacillus), though the presence of pathogenic bacteria (Staphylococcus and Salmonella) have been mentioned. The final microbial load depends upon the physical-chemical properties of each mayonnaise, essentially acidity and pH [2]. For this reason, international food regulations demand a minimum content of 0,2% in acetic acid and a pH below 4.2 in order to inhibit microbial growth. Water activity, which is usually low (around 0.93), also helps to maintain microbiological stability. Despite most manufactured mayonnaises are considered safe products, legislations also demand their microbiological control. This control essentially consists of enumeration of viable mesophilic aerobic bacteria, enumeration of viable enteric bacteria, and presence determination of Salmonella and Shigella, two pathogenic bacteria. The traditional method or assay employed for the above mentioned controls [3, 4] is the plating technique, which rely on growing microorganisms onto more or less specialized culture solid media. The advantages of plating are that is easy-to-use, cost-effective, and has been extensively evaluated; the drawbacks of plating are low speed and high labourintensive. Since the advent of the biotechnological revolution many other techniques have been developed to overcome these drawbacks and as alternative to culture, among them the so-called molecular techniques [5, 6]. The most popular molecular techniques are those based on genotyping, i.e. determining the identity of microorganisms based on its genetic material using a biological assay. The most widely used method for genotyping is Polymerase Chain Reaction (PCR), which consists of the specific amplification of a small portion of the genetic material of an organism in a complex sample, such as food, using thermostable DNA polymerses [7]. PCR has many advantages, such as simplicity, specificity, sensitivity, speed, cost-effectiveness, but its main drawback is that enumeration is very difficult to implement, then restricting it as to a detection method in the alimentary domain. A plethora of PCR-based assays have been published, though their use in food is limited. We have successfully used the PCR technique for the specific and sensitive detection of shigellae in mayonnaise [8]. We amplified a species-specific target, virA, a gene present in all virulent strains of Shigella. In an attempt to minimize test costs, we have developed a mutiplex PCR format, which means amplification of two targets in a single reaction, thus saving time in labour, and money in reagents and fungibles. In the mutiplex PCR format, we amplify virA and IS200, a insertion sequence present in most of Salmonella. Here we report the use of this multiplex PCR for the simultaneous detection of shigellae and salmonellae in mayonnaise.

1430

©FORMATEX 2010

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________

2. Material and methods The bacteria and yeast strains and their growth conditions are listed in Table 1. The precise composition of the nutritive broth I (NBI), nutritive broth II (NBII), and yeast broth (YEPD) were those stated by the Spanish Type Culture Collection (http://www.cect.org/). The Luria-Bertani (LB) was purchased from Gibco, and Buffered Peptone Water and Man-Rogosa-Sharpe (MRS) broths were purchased from Gibco and Merck, respectively. 2.1

DNA isolation

Bacteria were grown in broth overnight, sedimented, and lysed with detergent. DNA was extracted from lysed cells with phenol-chloroform (25:24, vol:vol) and precipitated with ethanol in the presence of sodium acetate [9]. Contaminating RNA was degraded by suspending DNA in 10 mM Tris-HCl (pH 8), 0.1 mM EDTA (pH 8), 1 µg/ml RNase A. Yeasts were grown in broth overnight, sedimented, lysed with a combination of detergent and Zymolase 20T (50 µg/ml, Sigma), and then DNA extracted from lysed cells as above described. 2.2

Polymerase chain reaction

Amplifications were done in 50-µl reaction mixtures, containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, 25 pmol of each primer, 1 U Taq polymerase (Biotools), and template DNA. Template was either isolated DNA or whole-bacteria in food samples. For single PCR either IS200 or virA couple of primers were used; for multiplex PCR both couples of primers were used. Cycling (35-times) conditions are as follows: Denaturation for 45 s at 94°C. Annealing for 30 s at 65°C. Polymerization for 30 s at 72°C. Primer sequences are: IS200-forward: 5'-GCC GAA GAT GAG TGT GTC GAG TT-3'. IS200-reverse: 5'-TTC GCC GTG TTC TTA CCC ACC GT-3'. VirA-forward: 5'-CTG CAT TCT GGC AAT CTC TTC ACA TC-3'. VirA-reverse: 5'-TGA TGA GCT AAC TTC GTA AGC CCT CC-3'. Amplifications products were detected, following 2% agarose gel electrophoresis, either by UV visualization or colour development. For this latter, DNA was transferred onto nylon membrane, and hybridised at high stringency with Dig-labelled probe, according to manufacturer’s protocol (Roche). The probe was detected using and anti-Dig antibody coupled with alkaline-phosphatase and nitroblue-tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates for colour development.

3. Results 3.1

Single-target PCR

Single amplifications either with IS200 or with virA primers gave amplicons of about 150 and 200 bp (Figure 1), respectively for salmonellae and shigellae, as expected. The exact nature of amplicons were demonstrated by sequencing amplicons obtained from S. thyphimurium HVM2 and S. dysenteriae CECT584. For IS200 primers, all tested strains of Salmonella but one gave amplifications (see Table 1). No false positives were detected. For virA primers, all tested strains of Shigella gave amplifications (see Table 1). Interestingly, all enteroinvaise E. coli gave amplifications with virA primers, which is consistent with the idea the EIEC and shigellae are the same pathovar [10]. In addition, no false positives were obtained with virA primers. Considering specificity as the ratio between true negatives and true negative plus false positives, both primers resulted on 100% specificity. In the same way, considering sensitivity as the ratio between true positives and true positives plus false negatives, 96.6% and 100% sensitivity were obtained respectively for IS200 and virA primers. 3.2

Single-target PCR in mayonnaise samples

We tested whether or not mayonnaise inhibit the PCR reaction. For this purpose, we used 5 µl of different mayonnaise solutions, either pure product or product diluted in buffered peptone water. We determined that a 1/10 dilution of mayonnaise is the minimum dilution factor to be used to avoid reaction inhibition for both IS200 and virA primers (not shown). 3.3

Multiplex PCR

Multiplex PCR, using as template DNA of both S. thyphimurium HVM2 and S. dysenteriae CECT584, gave two independent amplicons clearly discriminated after gel electrophoresis (Figure 2A). We sought at determining detection

©FORMATEX 2010

1431

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________

level for the multiplex format. For this purpose we used increasing amounts of DNA of S. thyphimurium HVM2 and S. dysenteriae CECT584, and determined level according to two methods. 25 and 10 fg of DNA were detected respectively by UV visualization (Figure 2A) or by probe hybridisation (Figure 2B). 3.4

Multiplex PCR for detection of whole cells in mayonnaise

We used mayonnaise samples containing different amounts of S. thyphimurium HVM2 and/or S. dysenteriae CECT584. For PCR, mayonnaise was diluted 1/10 in buffered water peptone. In this way, we determined that less than 10 cells can be successfully amplified and observed by simple UV visualization (Figure 3). As the maximum volume of solution used in PCR is 5 µl, the detection level is 2000 cells/ml (10 cells in 5 µl). 3.5

Implementing PCR for the detection of Salmonella/Shigella in mayonnaise

Implementation means to gather together legislation demands and PCR requirements. From one hand, legislations state that 25 g of mayonnaise must be devoid of Salmonella/Shigella. In the other hand, PCR requirements are minimum mayonnaise dilution factor (1/10), maximum sample volume (5 µl), and minimum detection level (2000 cells/ml). Considering the most unfavourable scenario, only one cell is present in 25 g of mayonnaise, we determined the minimum time needed to confidently applied the PCR method, i.e. the time needed by these pathogens to reach 2000 cells/ml. We then calculated generation time for S. thyphimurium HVM2 and S. dysenteriae CECT584 in 1/10 diluted mayonnaise at 37ºC without agitation. In these conditions, generation times were estimated to be 20 and 33 minutes respectively for Salmonella and Shigella. Therefore, minimum enrichment times to reach PCR detection level were 380 and 625 min., respectively for Salmonella and Shigella. To test the proposed method, we deliberately contaminated 5 g mayonnaise with either 12.3±2.4 cells of S. thyphimurium HVM2 or 13.2±3.1 cells of S. dysenteriae CECT584. The mayonnaise samples were diluted 1/10 in buffered peptone water, and incubated without agitation at 37ºC. With these cell densities it was expected to reach PCR detection level after 300 min. (5 h approximately) or 500 min. (8 hours approximately), respectively for Salmonella and Shigella. Therefore, aliquots of 5 µl were taken and analysed by PCR after the third hour of incubation. We obtained amplification with IS200 primers in the mayonnaise contaminated with Salmonella after the fourth hour of incubation (Figure 4A), and with virA primers in the mayonnaise contaminated with Shigella after the seventh hour of incubation (Figure 4B).

4. Discussion Administrations pay special attention to the quality and safety of commercialised food [11], reason by which they have developed special legislations on food control [12], for instance, microbiological control as an obligated practice in food industries. Salmonella and Shigella are two genera of bacteria, belonging to the family Entorobacteriaceae, that can be find naturally in the environment, animals and food [13]. These pathogens can cause in man severe, sometimes fatal, illnesses. Major outbreaks of these two human pathogens occur subsequent to population crowding, natural catastrophes, famine, or faecal contamination of waters and food. As the risk of salmonellae/shigellae contamination of food is not negligible, the presence of these pathogens in many foods, such as mayonnaise, must be controlled. Detection is carried out normally by the traditional culture technique, based on isolation and identification of the microorganisms in more or less specialized media. However, administrations and specially industries are demanding for new techniques bypassing the drawbacks commonly associated to the culture technique, as those mentioned in the Introduction Section. The advent of biotechnology, with the development of molecular biology techniques, has opened new doors to overcome these problems [14]. However, the application of these techniques for routine use on food industries is still challenging [15]. Among these techniques, PCR has been the most popular for the microbiological control [7]. One advantage of PCR over the culture technique is that the detection of two or more microorganisms can be done in a single assay. To our knowledge, this is the first paper reporting the use of a single-tube multiplex PCR assay for the detection of Salmonella and Shigella in mayonnaise. When evaluating PCR for the detection of microorganisms two important criteria must be satisfy: specificity and sensitivity [16]. Specificity considered as a way to detect false negatives and sensitivity as to detect false positives. The single PCR using the IS200 or virA primers resulted to be highly specificity and sensitive. The detection level is also an important criterion for evaluating the PCR [17]. Unfortunately, there is no standard for reporting the detection level and some authors indicate it as the grams of DNA or number of cells that can be detected. Due to absence of standard, both criteria were used. In one hand, as little as 10 fg of DNA can be detected. Considering that a typical bacteria carry 6 fg of DNA approximately, this amount of DNA would correspond to two cells. In the other hand, as little as 10 cells can be detected. These values are not far to that published elsewhere [8, 18, 19]. It is well known that different substances, among them food, can inhibit the PCR reaction [20]. For this reason, it was necessary to know the maximum volume of diluent that did not inhibit the PCR reaction. For this purpose, the bacterial

1432

©FORMATEX 2010

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________

strains were inoculated in mayonnaise and subsequently diluted in buffered peptone water. This diluent was used as it is commonly employed to enrich salmonellae and shigellae. The volume and dilution factor that did not inhibit PCR were 5 µl and 1/10, respectively. Altogether, these data served as to calculate detection level of the method: 2000 cells/ml. With this detection level it is obvious that the pathogens must be enriched in mayonnaise before being detected by PCR. Therefore, the major concern for the applicability of PCR for the detection of salmonellae/shigellae is that needs an enrichment step. Thus, the method has to be considered a hybrid between molecular and microbiological techniques. Despite of this, detection time with the PCR technique is shortest than the detection time with the traditional technique. This latter needs 3 days to be accomplished while the former would need, according to our estimations, only fourteen hours to be accomplished. Table 1 Strains used in this paper, its source and growth conditions, as well as results of PCR after agarose gel electrophoresis.

Strain

Source

PCR

Growth conditions (Broth/Temperature)

Alcaligenes denitrificans Alcaligenes faecalis Arthrobacter oxydans Arthrobacter sp. Bacillus sp. Enterobacter aerogenes Enterobacter faecalis Escherichia coli DH5α HB101 XL1-Blue O:1 O:124 (EIEC) O:28 (EIEC) O:111 (EPEC) O:127 (EPEC) Klebsiella pneumoniae Lactobacillus cellobiosus Lactobacillus sake Micrococcus luteus Mycobacterium phle Proteus vulgaris Pseudomonas fluorescens Salmonella S. abony S. agona S. blockey S. braenderup S. bredeney S. daressalaam S. dublin

S. infantis S. minnesota S. montevideo

CECT449 CECT145 CECT386 MY1A CECT450 MY1B CECT684 CECT481

NBI/26ºC NBI/37ºC NBII/26ºC PC/30ºC NBI/27ºC NBI/37ºC NBI/30ºC NBI/37ºC LB/37ºC

Gibco-BRL Gibco/BRL Stratagene CECT515 GP120 GP121 GP41 GP42 MDB171 MDE2348/69 CECT852 CECT562 CECT906 CECT241 CECT3009 CECT484 CECT378

NBI/37ºC MRS/30ºC MRS/30ºC NBI/30ºC NBI/37ºC NBI/37ºC BNII/26ºC LB/37ºC

CECT545 CECT705 HVM11 HVM40 CECT921 CECT700 CECT4000 HVM1 HVM29 HVM30 CECT700 CECT456 HVM10 HVM13 HVM15

©FORMATEX 2010

IS200 -

VirA -

-

+ + + + -

+ + + + + + + + + + + + + +

-

1433

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________

Table 1 continued.

Strain

Source

Growth conditions

PCR

(Broth/Temperature) HVM37 HVM46 Salmonella S. panama S. paratiphy A S. paratiphy B S. paratiphy C S. typhimurium

Serratia marcescens Shigella S. boydii

S. dysenteriae

S. flexneri

S. sonnei

Staphylococcus aureus Staphylococcus sp. Yersinia enterocolitica Yersinia pestis Cryptococcus sp. Pichia fermentans Rhodotorula sp. Saccharomyces cerevisiae Schizosaccharomyces pomber

+ + IS200

VirA

+ + + + + + + + -

+ + + + + + + + + + + + + -

LB/37ºC HVM20 HVM39 CECT825 CECT884 CECT699 HVM2 HVM12 HVM28 CECT159

NBII/26ºC LB/37ºC

CECT583 GP26 GP238 CECT584 GP193 GP300 CECT585 GP295 GP299 CECT457 CECT542 GP296 GP298 CECT240 MY1S GK EC MI CECT1455 MI MI

NBI/37ºC PC/37ºC NBI/37ºC NBI/28ºC YEPD/24ºC YEPD/24ºC YEPD/24ºC YEPD/24ºC

-

MI

YEPD/24ºC

-

Abreviations: CECT: Colección Española de Cultivos Tipo; MY: mayonesa Ybarra isolated; GP: kindly donated by G. Prats, Hospital Universitario Sant Creu i Sant Pau, Barcelona, Spain; MD: kindly donated by M. Donnenberg, University of Maryland School of Medicine, Baltimore, USA; HVM: kindly donated by JC Palomares, Hospital Universitario Virgen Macarena, Sevilla, Spain; GK: kindly donated by G. Kapperud, National institute of Public Health, Oslo, Norway; EC: kindly donated by E. Carniel, Institut Pasteur, Paris, France; MI: kindly donated by Departamento de Microbiología, Universidad de Sevilla, Sevilla, Spain; EIEC: Enteroinvasive Eschechia coli; EPEC: Enteropathogenic Eschechia coli.

1434

©FORMATEX 2010

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________

Fig. 1 Agarose gel electrophoresis showing single PCR amplifications. Genomic DNA from Salmonella thyphimurium HVM2 or from Shigella dysenteriae CECT584 were used as templates for PCR either with IS200 or with virA primers, respectively. As expected IS200 amplification results in an amplicon of about 150 bp (lane 1) while virA amplification results in an amplicon of about 200 bp (lane 2). Lane 3 corresponds to a 100-bp size marker.

Fig. 2 Agarose gel electrophoresis (A) and hybridisation (B) showing multiplex PCR amplifications. Different amount of genomic DNA from Salmonella thyphimurium HVM2 and Shigella dysenteriae CECT584 were used as templates for PCR with IS200 and with virA primers. The hybridisation in B corresponds to the same gel than in A. Lanes 1-6: 0.5, 1, 10, 25, 50, and 100 fg of both bacterial DNAs. Lane 7: 100-bp size marker.

Fig. 3 Agarose gel electrophoresis showing multiplex PCR amplifications. Different amount of cells from Salmonella thyphimurium HVM2 and/or Shigella dysenteriae CECT584 were used as templates for PCR with both IS200 and with virA primers. Lane 1: 4 cells of Shigella. Lanes 2: 4 cells of Salmonella. Lanes 3: 6 and 4 cells of Shigella and Salmonella. Lane 4: 62 and 43 cells of Shigella and Salmonella. Lane 5: a 100-bp size marker.

©FORMATEX 2010

1435

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology A. Méndez-Vilas (Ed.) _______________________________________________________________________________________

Fig. 4 Agarose gel electrophoresis showing single PCR amplifications after different times of enrichment. Samples of 1/10 diluted mayonnaise where deliberately contaminated with Salmonella thyphimurium HVM2 or Shigella dysenteriae CECT584 and incubated at 37ºC without agitation. After 3 hours of enrichment, a 5 µl sample was taken and analyse by PCR with IS200 (panel A) or virA (panel B). (A) Lanes 1-4: 3, 4, 5, and 6 hours of enrichment respectively; Lane 5: a 100-bp size marker. (B) Lanes 1-3: 6, 7, and 8 hours of enrichment respectively; Lane 4: a 100-bp size marker. Acknowledgements The support by Ministerio de Ciencia y Tecnología (grants BOS2003-01072, BFU2009-10393, and fellowships) and Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (grants to BIO-210 group) are gratefully acknowledged. Author is also gratefully acknowledged to E. Trujillo and J. L. Algeciras for providing Ybarra mayonnaise, to J.C. Palomares, E. Carniel, M. Donnenberg, G. Kapperud and G. Prats for providing bacterial strains, and to Antonio Torres for fruitful discussions.

References [1] Smittle RB: Microbiology of mayonnaise and salad dressing: a review. Journal Food Protection 1977; 40:415-422. [2] Silliker JH, Elliott RP, Baird-Parker AC, Bryan FL, Christian JHB, Clark DS, Olson JC, Roberts TA Jr. Microbial ecology of foods vol 2. Factors affecting death of microorganisms. Academic Press; 1980. [3] de Boer E, Beumer RR. Methodology for detection and typing of foodborne microorganisms. International Journal Food Microbiology. 1999;50:119-30. [4] Jay JM, Loessner MJ, Golden DA. Modern food microbiology. 7th ed. Springer Science; 2005. [5] Iqbald SS, Mayoa MW, Brunoa JG, Bronkb BV, Battc CA, Chambers JP. A review of molecular recognition technologies for detection of biological threat agents. Biosensors and Bioelectronics. 2000;15:549-578. [6] Velusamya V, Arshaka K, Korostynskaa O, Oliwab K, Adleyb C. An overview of foodborne pathogen detection: in the perspective of biosensors. Biotechnology Advances. 2010;28:232-254. [7] Lantz PG, al Soud WA, Knutsson R, Hahn-Hagerdal B, Radström P. Biotechnical use of polymerase chain reaction for microbiological analysis of biological samples. Biotechnology Annual Reviews. 2000;5:87–130. [8] Villalobo E, Torres A. PCR for detection of Shigella spp. in mayonnaise. Applied Environmental Microbiology. 1998; 64:12421245. [9] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. 2nd ed. Cold Spring Habor Laboratory Press; 1989 [10] Penga J, Yanga J, Jin Q. The molecular evolutionary history of Shigella spp. and enteroinvasive Escherichia coli. Infection Genetics Evolution. 2009;9:147-152. [11] White Paper on Food Safety. Commission of the European Community. Available at: http://ec.europa.eu/dgs/health_consumer/library/pub/pub06_en.pdf. Accessed May 31, 2010. [12] Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. Available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2002:031:0001:0024:EN:PDF. Accessed May 31, 2010. [13] Doyle M. P. Foodborne bacterial pathogens. Marcel-Dekker; 1989. [14] Stewart. GSAB. Challenging food microbiology from a molecular perspective. Microbiology. 1997;143:2099-2108. [15] Rodríguez-Lázaro D, Cook N, D'Agostino M, Hernández M. Current challenges in molecular diagnostics in food microbiology. Global Issues in Food Science and Technology; 2009: 211-228 [16] Malorny B, Tassios PT, Radström P, Cook N, Wagner M, Hoorfar J. Standardization of diagnostic PCR for the detection of foodborne pathogens. International Journal Food Microbiology. 2003;83:39–48. [17] Markert-Hahn C, Berding C, Kuhn C. Critical level and detection limit: performance measures for PCR-based assays. Journal Virological Methods. 1998;74:139–148. [18] Hill WE: The polymerase chain reaction: applications for the detection of foodborne pathogens. Critical Reviews Food Science Nutrition. 1996;36:123-173. [19] Bej AB, Steffan RJ, DiCesare J, Haff L, Atlas RM. Detection of coliform bacteria by polymerase chain reaction and gene probes. Applied Environmental Microbiology. 1990;56:307-314. [20] Rossen L, Norskov P, Holmstrøm K, Rasmussen OF. Inhibition of PCR by componets of food samples, microbial diagnostic assays and DNA-extraction solutions. International Journal Food Microbiology. 1992;17:37-45.

1436

©FORMATEX 2010