comparison of specificity of different primer pairs for ...web.firat.edu.tr/ijst/3-2/(1) senan...

International Journal of Science & Technology Volume 3, No 2, 123- 137, 2008

Comparison of Specificity of Different Primer Pairs for The Development of Multiplex PCR Assays for Rapid Identification of

Dairy Lactobacilli

Suja Senan*, Sunita Grover**and Virender Kumar Batish**

*Department of Dairy Microbiology, SMC College of Dairy Science, Anand Agricultural University, Anand – 388 110, Gujarat, INDIA.

** Molecular Biology Unit, Dairy Microbiology Division, National Dairy Research Institute, Karnal-132001, Haryana, INDIA.

[email protected](Received: 14.05.2008; Accepted: 20.08.2008)

Abstract:This work focused on the (1) comparison of the specificity of 4 conserved and 11 specific primer pairs derived from the integrated sequences of Lactobacilli 16S and 23S ribosomal RNA genes and their intergenic spacer region (2) designing multiplex PCR assays for the identification of Lactobacillus species. Fifty three reference strains, 21 closely related genera of lactobacilli, and 36 Lactobacillus isolates were subjected to genus specific PCR reactions. Confirmed Lactobacilli were subjected to species specific PCR assays for Lactobacillus acidophilus, Lb. helveticus, Lb. plantarum, Lb. casei, Lb. fermentum, Lb. delbrueckii, and Lb. rhamnosus. We encountered certain ambiguous results for the PCR assays when applied to several Lactobacillus strains, hence lay stress on the need for great caution in routine analysis of dairy lactobacilli. Multiplex PCR assays were further developed by combining two, three or four sets of primers tested previously. Out of the 16 multiplex reactions, 3 reactions showed optimum clarity and reproducibility. The successful reactions were specific for the identification of (a) Lb. fermentum and Lb. plantarum (b) Lb. casei and Lb. rhamnosus and (c) Lb. delbrueckii and Lb. rhamnosus. The developed multiplex PCR assay can act as a rapid tool to differentiate closely related Lactobacilli with different functionality.

KEY WORDS: Lactobacilli, identification, primers, multiplex PCR

1. Introduction

Lactobacilli have found its application in areas like fermented foods, silages, fish and crab waste management and poultry by product industries. Several strains of Lactobacilli (Lactobacillus rhamnosus GG, Lb. casei Shirota, Lb. paracasei CRL431, Lb. fermentum RC14) have been associated with the development of probiotic dairy foods. Hence Lactobacilli have been generating a great deal of consumer, industrial and scientific interests. Acknowledging the fact that these technological and therapeutic attributes are strain or species dependent, it is evident that there is a need for reliable and sensitive identification techniques for definitive analysis of the relevant strains in the fermentation product and probiotic strain integrity. Phenotypic methods with limited taxonomic resolution and ambiguous results are inadequate for the identification of lactobacillus species [Dellaglio and Felis [1]].

Molecular techniques have improved the discriminatory power of identification. Polymerase Chain Reaction (PCR) finds wide application in detection of lactobacilli [Giraffa et al. [2]]. Integral to the efficacy of a PCR reaction is the choice of primers and the target gene. Among the target genes, the 16S rRNA gene sequence diversity has been widely validated as a means of identification for lactobacilli at genus and species level [for example, Nakagawa et al. [3], Drake et al. [4], Quere et al. [5], Andrighetto et al. [6], Berthier and Ehrlich [7], Ward and Timmins [8], Torriani et al. [9], Walter et al. [10], Yost et al. [11], Muller et al. [12], Song et al. [13], Chagnaud et al. [14], Berthier et al. [15], Heilig et al. [16], Dubernet et al. [17], and Couret et al. [18]]. With the increasing number of primers, the manufacturer of a probiotic supplement or a probiotic dairy product is faced with the challenge of selecting the most suitable primer set. Comparing different primers for their discriminatory power is a

mailto:[email protected]

S. Senan, S. Grover, V. Kumar Batish

124

reliable way to identify the most promising primer set before applying them for routine analysis. Applying the published primers and the suggested PCR conditions directly, without prior validation can lead to discrepancies in results, affecting the reliability and reproducibility of the proposed assays. Ikonomopoulus et al. [19] indicated that the type of thermocycler and the type of polymerase enzyme incorporated to the PCR assay can have a significant impact on the performance of a reaction. Other factors like the genetic diversity of strains analysed, presence of PCR inhibitors and DNA fragmentation can lead to false positive and false negative results leading to discrepancies in interlaboratory results. In the present study, we evaluated the specificity of 4 conserved and 11 specific primer pairs using 53 reference strains of lactobacilli and 21 closely related genera. Apart form the reference strains, the standardized PCR assays were also applied to 36 strains isolated from environmental samples such as human milk, cow milk, infant faeces and commercially available probiotic preparations. This gave the study a four fold advantage of (1) revealing the taxonomic characteristics of environmental isolates (2) identifying novel functional strains (3) checking the reproducibility of standardized PCR assays and developed multiplex PCR reactions and (4) confirming the identity of strains claimed on the labels of commercial probiotic preparations. The selected primers displaying satisfactory specificity were used for the development of multiplex PCR assays. The cultures put to test for the development for multiplex PCR reactions included each from the three groups of lactobacilli cultures. (i) Obligately homofermentative lactobacilli (Lb. delbrueckii ssp. lactis; Lb. delbrueckii ssp bulgaricus) (ii) Facultative heterofermentative lactobacilli (Lb. casei, Lb. rhamnsosus, Lb. plantarum) (iii) Obligately heterofermentative lactobacilli (Lb. fermentum).

2. Materials and Methods:

2.1 Reference strains and growth conditions

A collection of 53 reference strains of Lactobacilli, 15 non-lactobacilli and 6 non-lactic acid bacteria used in this study were obtained from National Collection of Dairy Cultures (NCDC) and Molecular Biology Unit (MBU), National Dairy Research Institute, Karnal, Haryana, India and are listed in Table 1. Bacteria requiring anaerobiosis for growth were cultured at 37°C in an anaerobic chamber (MK III Anaerobic Work Station, Don Whitley, UK; 80% Nitrogen, 10% Hydrogen and 10% Carbon dioxide). Man–Rogosa-Sharpe (MRS) broth [De Man et al. [20]], M-17 broth [Terzaghi and Sandine [21]], modified MRS medium with 0.05% cysteine hydrochloride and Brain Heart Infusion broth (Hi Media lab., Mumbai, India) were used for the selective culturing of Lactobacillus spp. ; Lactococci, Leuconostoc spp.; Bifidobacterium spp. and other bacteria respectively. The purity of the bacterial cultures were monitored by Gram staining. 2.2 Bacterial strains, isolation and viability

A total of 36 Lactobacillus strains were isolated from 5 raw cow milk samples, 12 unprocessed human milk samples, 2 infant faecal samples, and 7 probiotic preparations. The cow milk and human milk samples were collected aseptically by following standard methods [AOAC, [22]]. Freshly voided infant faeces were collected at home by the parents and put in a sterile plastic bag and kept refrigerated until transportation to the laboratory, where they were analyzed within 2 hours after collection. The probiotic supplements were purchased from the market and the recommended storage conditions were maintained. They were analyzed before the end of their expiry period. All the samples were serially diluted and plated on MRS agar at 37°C for 16 - 18 h for selective outgrowth of lactobacilli. All strains grew well without the use of anaerobic conditions. From appropriate dilutions one representative colony was picked and tentatively identified as lactobacilli after Gram stain reaction, colony appearance, cell morphology and carbohydrate fermentation patterns as delineated by Bergey’s manual [Hensyl [23]].

Comparison of Specificity of Different Primer Pairs for The Development of Multiplex PCR Assays

125

2.3 Isolation of DNA The genomic DNA from all strains was

extracted by following standard protocol [Pospiech and Neumann [24]]. DNA concentration was determined by absorbance at 260 nm in a UV Spectrophotometer (DU640, Beckman, USA). The purity of DNA was ascertained by measuring OD ratios at 260 to 280 nm and integrity of DNA was tested by running on 1% agarose gel electrophoresis [Sambrook et al. [25]]. 2.4 Selection of primers

For Lactobacillus specific primers, we tested universal primers SS1/SS2 [Rochelle et al. [26]] and 16/23-10C [Song et al [13]] targeted against different conserved regions of 16S/23S rRNA for establishing the Eubacterial origin of the cultures. Semi-universal primers Lb1/Lb2 [Klijn et al. [27]] based on a variable loop in 16S rRNA sequence of Lactobacilli species and primers designed by Dubernet et al. [17] which included 21 mer primer LbLMA-1 (lactobacilli specific sequence) coupled with a 21 mer universal sequence R16-1 from the flanking terminal region of the 16S rRNA gene were then applied to determine the genera of the type strains. Species specific primers, CA1 and DB1 [Drake et al. [4]] for Lactobacillus casei and Lactobacillus delbrueckii subsp. bulgaricus / lactis respectively were investigated in this study. These primers were designed on the basis of the sequences obtained from the Ribosomal Database Project (University of Illinois, Urbana, USA). Each species specific primer was used in conjunction with appropriate universal primer SS1 or SS2 to obtain species specific amplification from rRNA sequences. Apart from these, species specific primers designed by Song et al [13] targeted against 16S-23S rRNA interspacer region and its flanking 23S rRNA were also evaluated and compared with those of Drake to further correlate PCR results. The sequence and the amplicon size of the primers used in this study are listed in Table 2.

2.5 PCR conditions:

Oligonucleotide primers used for the PCR were synthesized by Bangalore Genei (Bangalore, India). The polymerase chain

reactions (25 µl) contained 10 pmol of each primer, 0.2mM of each dNTP, 1X PCR buffer and 0.5 IU of Taq polymerase (Bangalore Genei, Bangalore, India) with 1 µl of template DNA. The reaction was carried out in a thermal cycler (Eppendorf master cycler gradient, 5331, Germany). The PCR assay was performed following the published PCR conditions [[4], [5], [13] and [17]] with appropriate modifications when discrepancies in results were encountered. Appropriate positive and negative controls were also set up along with the test samples to assess the efficacy of the PCR reagents. Amplified fragments were separated by agarose gel electrophoresis (1 % w/v) (DNA subcell, BioRad, USA) in 1X TAE buffer, or non denaturing SDS- Polyacrylamide gel electrophoresis( 5 %). The gels were visualized by staining with ethidium bromide (0.5 µg/ml) and photographed under UV Transilluminator using Polaroid Camera (Fotodyne, USA). 3. Results and Discussion 3.1 Specificity of Lactobacillus genus specific primers

PCR assay conditions using universal primers SS1/SS2 were followed as per the published protocol. However, all non-Lactobacillus cultures, i.e., Lactococcus sp., Pediococcus sp., Leuconostoc sp., Bifidobacterium sp., Propionibacterium species including Gram-negative E. coli gave positive signals These results substantiate the highly conserved nature of the sequences of the rRNA against which primers were designed and hence their ineffectiveness to be used as a Lactobacillus genus specific primer. Further, we evaluated the universal primers, 16/23-10C to establish the identity of the cultures as eubacteria. Our results in this regard are comparable to those obtained by Song et al. [13] (data not shown) that all the test cultures showed an amplified product of 600 bp, thereby, indicating their eubacterial origin but lacked specificity against closely related genera. PCR assay using the third set Lb1/Lb2 was positive for all the 53 reference cultures of Lactobacilli species resulting in the amplification of a 194 bp product, which was in accordance with the literature. However, this


126

PCR assay showed some cross reactivity when examined with related genera like Pediococcus, Leuconostoc and Bifidobacterium species (data not shown). As the primers were designed from the variable loop in 16S rRNA sequence of Lactobacillus, no cross reactivity should have been observed with template DNA from non-lactobacilli. The false positive result obtained with this primer set in our study was similar to the observations of Weiss et al. [28]. Hence, the aforementioned primers were not considered reliable due to their low selectivity for the genus Lactobacillus. For the evaluation of set 4 primers LbLMA-1/ R16-1, PCR conditions were standardized with respect to the annealing temperature By subjecting a reference Lactobacillus strain to gradient PCR we obtained the best amplications at 59°C compared to 55°C as suggested in the literature. This discrepancy in results could be attributed to minor differences in PCR reaction set ups including the quality of PCR reagents, template DNA, and design of PCR machine used in different laboratories. The PCR assay produced an amplified product of 250 bp with all lactobacillus reference strains tested in this study. The results obtained from some representative Lactobacillus species and isolates from raw cow milk are shown in Fig 1. This primer pair was judged for their specificity against Pediococcus pentosaceus, Leuconostoc mesenteroides, Propionibacterium freudenreichii subsp shermanii, Lactococcus lactis subsp lactis and Lactococcus lactis subsp cremoris However, 250 bp amplified product could not be detected with any of the aforesaid cultures. Higher selectivity for Lactobacilli was observed for the primer pair LbLMA-1/ R16-1 and was declared most suitable to be used for confirming Lactobacillus genus affiliation. 3.2 Specificity of Lactobacillus species grouping primers

Since lactobacilli represent an extremely heterogeneous group, it was appropriate to first assign them into various groups. This was achieved by exploiting the similarities and dissimilarities in the 16S-23S rRNA ISR (intergenic spacer region) and flanking 23S

rRNA. We evaluated group specific primers, namely Ldel-7/Lac 2 (Group I: Lb. delbrueckii ssp. bulgaricus / lactis), Lu1/Lac2 (Group II: Lb. acidophilus, Lb. helveticus, Lb. amylovorus, Lb. crispatus, Lb. gasseri, Lb. johnsonii), Lu-5/Lac2 (Group III: Lb. paracasei, Lb. casei and Lb. rhamnosus.), Lu-3/Lac2 (Group IV: Lb. plantarum and Lb, fermentum) for allocating lactobacilli species used in our study. As shown in Fig 2., out of the seven Lactobacillus acidophilus and five Lactobacillus helveticus cultures (Gr. II) only two cultures (La11 and La195) (Lanes: 9 and 10) gave a positive result as depicted by the specific amplicon of 300 bp. Invariably an additional band of 700 bp appeared in all the cultures. These results are at variance with Song’s data that performed their experiment with a limited number of cultures. We also observed non specific bands of 700 bp and and 350 bp in case of Group III primer set when we tested it against 6 Lb. casei, 2 Lb. paracasei and 4 Lb. rhamnosus. The presence of non specific bands call for additional stringency in the PCR primers and further improvement in the PCR assay .Group IV primer gave the expected band of 350 bp with all the twelve Lb. plantarum and Lb. fermentum cultures used in the study. Group I comprised of Lb. delbrueckii subsp. bulgaricus/lactis and the results will be discussed subsequently in section 3.3.3.

3.3 Specificity of Lactobacillus species

specific primers 3.3.1 Lactobacillus acidophilus and

Lactobacillus helveticus For this study, we included seven strains of Lb. acidophilus and 5 strains of Lb. helveticus as templates. A 1400 bp amplified product specific for Lb. acidophilus was observed with only two of Lb. acidophilus cultures, namely La11 and La195 and one of Lb. helveticus Lh 288. However, due to the same size of amplicon, i.e., 1400 bp, the 2 species of Lb. acidophilus and one species of Lb. helveticus could not be differentiated on agarose gel due to low variability in their rRNA sequence (less than 3%). However, the two species could be delineated by EcoR1 digestion of the amplicon


127

that was cleaved into two fragments (800 and 600 bp) only in case of Lb. helveticus (Fig.3, lane 6). The specificity of PCR assay was also tested by applying the assay to selected non-targeted Lactobacillus species, such as Lb. casei and Lb. delbrueckii subsp. bulgaricus (data not shown). None of these non targeted lactobacilli gave positive signal with the assay, thus, indicating the specificity of the primers in the test. Our results in this regard are consistent with observations of Drake et. al [4]. The identity of Lb. acidophilus was further confirmed with the help of primers Laci-1/23-10C [Song et al. [13]]. Lb. acidophilus strains (La11, La195) gave a positive response and showed the specific amplified products of 210 bp thus, confirming them as Lb. acidophilus. These results are exactly comparable to those obtained by following PCR protocol developed by Drake as explained previously. 3.3.2 Evaluation of primers for the identification of Lactobacillus casei

In this study, the efficacy of the primers SS1/CA1 Drake et. al [4], and Lu5/Lac2 [Song et al. [13]] specific for Lb. casei group was tested by applying the PCR assay on five reference cultures of Lb. casei listed in Table 1. All the five Lb. casei cultures produced 1200 bp product specific for Lb. casei (SS1/CA1) and in accordance with the published literature. The result of primer set 14 was discussed earlier in 3.2. Based on the reproducibility of our results, it can be concluded that the PCR assays based on the primer pair SS1/CA1 are suitable for identification of Lb. casei and can be extended for applying them in multiplexing for the identification of Lb. casei. 3.3.3 Evaluation of primers for the identification of Lactobacillus delbrueckii subsp. bulgaricus / lactis

For identification of Lb. delbrueckii subsp. bulgaricus/ lactis, the species specific primers SS1/DB1 Drake et. al [4] were explored. The PCR assay was applied to all the 13 reference strains of Lb. delbrueckii subsp. bulgaricus and 4 Lb. delbrueckii subsp. lactis to confirm their identity. The results pertaining to the same are shown in gel pictures (Fig. 4). A critical appraisal of PCR amplified bands on

agarose gel revealed an unamibiguous appearance of either 1200 bp product or 1400 bp. Polymorphisms of the amplicons were observed in accordance with the literature Drake et. al [4]. Surprisingly in this study, we could also detect the presence of both 1200 bp & 1400 bp products with some of the targeted cultures (Ldb184; Ldb308). Such an observation was not reported in literature. This discrepancy in results could be attributed to comparatively few strains of Lb. delbrueckii used in the published literature (5 Lb. delbrueckii subsp. bulgaricus and one Lb. delbrueckii subsp. lactis). Another drawback of this set of primers is their inability to discriminate between both subspecies of Lb. delbrueckii. When PCR results of this study were analysed critically, it was observed that when both the products were amplified together, there was a tendency of enhanced amplification of 1400 bp product. At the moment, we are not in a position to explain this peculiar situation due to lack of any published report on these lines. In order to corroborate the results obtained with SS1/DB1 specific for Lb.delbrueckii, an attempt was also made to identify the aforesaid cultures by PCR using additional primer pair Ldel7/Lac2 [Song et al. [13]]. The species Lb delbrueckii produced an amplified product of 450 bp. An additional 800 bp product was invariably observed in most of the cultures as indicated in Fig. 5. Among the entire test Lb. delbrueckii strains used in the study, 7 cultures gave bands of 450 bp and 800 bp, unambiguously, thereby, establishing their identity as Lactobacillus delbrueckii subsp. bulgaricus or Lactobacillus delbrueckii subsp. lactis. The identity of these eight cultures is further supported by the results obtained from using primer pair SSI/DBI. Interestingly, the identity of five cultures (Ldb253, Ldb 281, Ldb 304, Ldl 308) which were identified as Lactobacillus delbrueckii based on Drake's primers could not be substantiated by the aforesaid Ldel7/Lac2 primers. The possible reasons for lack of positive correlation between the two PCR results with respect to the aforesaid cultures are subject to speculations. 3.3.4 Evaluation of primers for the identification of Lactobacillus rhamnosus


128

When the four Lb. rhamnosus strains (Lr18, Lr19, Lr24, LrMBU3) were tested with the PCR assay using Lb. rhamnosus specific primers Lu5/RhaII in this study, all the cultures produced desired 113 bp band specific for Lb. rhamnosus. These results indicate that the four cultures were undoubtedly Lb. rhamnosus (data not shown). 3.3.5 Evaluation of primers for the identification of Lactobacillus fermentum

For confirming identity of Lactobacillus fermentum with the PCR assay using Lactobacillus fermentum specific primers, viz., Lfer3/Lfer 4, we were able to unequivocally confirm that three out of six Lb. fermentum cultures used in the study were genuine Lb. fermentum as revealed by the formation of 192 bp product (Fig. 6). These results were obtained after optimizing the PCR amplification conditions by running a gradient PCR (58 to 62°C) wherein the best amplification was achieved at 62°C. However, Song et al. (2000) had reported the best results at an annealing temperature of 60°C.

3.3.6 Evaluation of primers for the identification of Lactobacillus plantarum

In our study, we used six Lactobacillus plantarum, for confirming their identity by PCR assay using primers Lpla2/Lpla3 (Song et al., 2000). Results pertaining to this study are depicted in Fig. 6. As evident from gel pictures, four of the cultures (Lpla1, Lpla20, Lpla21 and Lpla221) showed a distinct 248 bp product specific for Lb. plantarum, thereby suggesting these cultures as true Lb. plantarum strains. 3.3.7 Identification of wild lactobacilli isolates at genus and species level

After evaluating the specificity of the primers, they were applied to 36 Lactobacillus isolates for their identification at genus and species level. They were subjected to the genus specific PCR assay using primers LbLMA-1/R16-1 along with a reference Lactobacillus culture as a positive control. All the 36 isolates gave an amplified product of 250 bp and were

characterized as lactobacilli (Fig 1, Lane 5, 10). The majority of the isolates from probiotic supplements evaluated in this study failed to amplify the Lactobacillus specific amplicon although their labels contain Lactobacillus sporogenes. The non amplification could be due to misleading nomenclature since this species has been classified as Bacillus coagulans now. Similar results were also observed by other authors De Vecchi and Drago [29]. The presence of Bacillus species was further confirmed by phenotypic tests like catalase and presence of spores by staining.

All the isolates identified as Lactobacilli were subjected to PCR assays using species specific primers, SS1/CA1, SS1/DB1 and SS2/HE1. Four isolates of probiotic preparations were identified as Lb. casei (Data not shown). Although Lb.casei was not listed as an ingredient on the labels of the probiotic preparation, its presence may represent contamination of the samples related to the fact that manufacturers utilize shared equipment to produce all probiotics and PCR is a highly sensitive technique [Drisko et. al. [30]. Three of the cow milk isolates also produced a distinct band of 1200 bp product and were identified as Lb. casei. However, the species identity of the human isolates could not be determined as no PCR product could be obtained with any of the aforesaid three sets of primers. Hence, it might belong to lactobacilli species other than Lb. casei, Lb. delbrueckii and Lb acidophilus/ Lb helveticus. Similarly, out of the eight isolates obtained from faecal samples which showed 250 bp product in the Lactobacillus genus specific assay, five isolates gave a 1200 bp product only with SS1/DB1 based PCR assay only, thereby, establishing their possible identity as Lb. delbrueckii.

3.4 Development of Multiplex PCR for the Identification of Lactobacillus Species

Efforts were then made to develop multiplex PCR by combining two, three or four sets of primers designed by Drake et al. [4] and Song et al. [13] tested previously in their


129

respective conventional PCR assays, based on their compatibility and amplified product size for rapid identification of different Lactobacillus species in one reaction. The careful analysis of the gel profiles (Figs. 7 and 8) of 16 multiplex PCR reveal that specific and reproducible amplification of the specific products against all the target species was achieved with only three of the multiplex PCR assays developed in this study, which was specific for the identification of Lb. fermentum, Lb. plantarum; Lb. casei, Lb. rhmanosus; and Lb. delbrueckii , Lb. rhamnosus, respectively. In case of MP-8 (Fig 7, Lane 8) all the 3 bands of expected size, viz. 1400 bp (specific for eubacteria), 248 bp (Lpla2/Lpla3) and 192 bp (Lfer3/Lfer4) could be observed. Similarly, in M-14 (Fig 8, Lane3), the two specific amplified products of 1200 bp (SS1/CA1) and 113 bp (Lu5/RhaII) were clearly discernible which were specific for Lb. casei and Lb. rhmanosus, respectively. Almost a similar trend was observed with M-15 (Fig 8, Lane4), wherein only two specific bands of expected size of 450 bp (Ldel7/Lac2) and 113 bp (Lu5/RhaII, specific for Lb. rhmanosus) could be detected. The low success rate can be attributed to the difficulty in optimizing PCR conditions for large number of primer pairs and insufficient priming targets due to low variation in the rRNA gene which reflects its conservation all through the evolution. 4.0 Conclusion:

In view of the enormous value of lactobacilli as starters and probiotics in dairy industry, their reliable and rapid identification by PCR assays is extremely important. Knowledge of the performance of commercially available primers, reagents and enzymes are critical for the careful designing of analytical PCR assay systems in Quality Assurance departments of food and dairy industry. The success of PCR assays for reliable identification of lactobacilli both at genus and species level is chiefly dependent

upon the designing of the primers targeted against specific genes. This study provides important comparative data on specificities of the primers that are currently being used in different food analysis labs all over India for the identification of functional lactobacilli. In this study we faithfully adhered to the recommended protocols published for each of the fifteen assays with respect to the experimental conditions in our laboratory settings. On comparing the four genus specific primers, LbLMA1/R 16 -1 was highly specific to Lactobacillus in our study stystem.

With regard to the target specificity of species specific primer sets analysed in this study, Primer set SS2/HE, Lfer3/Lfer4, Lpla2/Lpla3, and Lu5/RhaII showed a higher degree of specificity. However, primer sets other than the above mentioned showed non specific products in multiple numbers and cross reactivity with the strains studied. The reasons for such ambiguity between the published results and the present study could be the use of different brand of PCR enzymes, buffers and thermocyclers manufactured in various corners of the world that can affect the efficacy of the reaction. The wide variation in the strains studied in matters of taxonomical diversity and test numbers could also lead to discrepancies. We eliminated the probable effect of DNA isolation protocol on the amplification efficiency of PCR assays by following a standard procedure for each strain. Hence, standardization of PCR assays after thorough evaluation of specific primers against as many cultures as possible offers a realistic safe gaurd to rule out the possibility of false negative and false positive results. We conclude by laying emphasis on two points, (1) the need for great caution in interpreting the results in routine analysis of probiotic and dairy lactobacilli and (2) the need to harmonize the parameters of a PCR reaction to avoid interlaboratory discrepancies in results, especially among countries.


130


131


132


133

Table 1. List of Lactobacillus reference strains used in the present study.

Genus-species-subspecies Stain designation Reference strains Lactobacillus acidophilus La11, La13, La14, La15, La16, La195, La291 Lactobacillus helveticus Lh5, Lh194, Lh288, Lh292, LhMBU11 Lactobacillus casei Lc298, Lc299, LcMBU7, LcMBU9, LcMBU13

Lactobacillus delbreuckii subsp. bulgaricus Ldb4, Ldb9, Ldb26, Ldb184, Ldb253, Ldb277, Ldb281, Ldb285, Ldb293, Ldb304, Ldb308, LdbMBU2

Lactobacillus delbreuckii subsp. lactis Ldl3, Ldl10, Ldl283, Ldl290 Lactobacillus fermentum Lf141, Lf155, Lf156, Lf214,LfMBU5, LfMBU8

Lactobacillus paracasei subsp. paracasei Lpar 63, LparMBU4

Lactobacillus plantarum Lpl20, Lpl21, Lpl25, Lpl201, Lpl221, LplMBU1

Lactobacillus rhamnosus Lr18, Lr24, Lr19, LrMBU3 Lactobacillus kefir LkefMBU10 Lactococcus lactis subsp.diacetylactis Lcd 60, Lcd 61, Lcd 81, Lcd 87

Lactococcus lactis subsp.cremoris Lcc 81

Lactococcus lactis subsp.lactis Lcl87

Pediococcus pentosaceus Pdb38, Pd35

Propionibacterium freudenreichii subsp shermanii Pr139, Pr140

Leuconostoc mesenteroides subsp. dextranicum Lem29

Leuconostoc lactis Lel200

Bifidobacterium lactis BifMBU2, BifMBU3, BifMBU5

Salmonella typhi SalMBU12

Shigella flexneri ShiMBU56

E. coli EcMBU8

Listeria monocytogenes LiMBU4

Yersinia enterocolitica YeMBU51

Bacillus cereus BcMBU22


134

Table 2. List of

Lactobacillus

reference strains used in

the present study

Primer Sets Sequence

Amplicon (bp)

Reference

Lactobacillus genus specific primers Set 1

SSI SS2

g tg c tg cag aga g t t t ga t cc tgg c t c ag cac gga t cc t ac ggg t ac c t t g t t acg ac t t 1400 [26]

Set 2

16 23-10C

gc t gga t ca cc t t t c gc t t t g cc t cac gg t ac t g

600 [13]

Set 3

Lb1 Lb2

aga g t t t ga t ca tgg c t c ag cgg t a t t ag ca t c tg t t t cc

194 [27]

Set 4

LbLMA1 R 16 - 1

c t c aaa ac t aaa caa ag t t t c c t t g t a cac acc gcc cg t ca 250 [17]

Lactobacillus species specific primers Set 5

SS1 CA1

gtg ctg cag aga gtt tga tcc tgg ctc ag tga tct ctc agg tga tca aaa 1200 [4]

Set 6

SS2 HE1

cac gga tcc tac ggg tac ctt gtt acg act t agc aga tcg cat gat cag ct 1400 [4]

Set 7 SS1 gtg ctg cag aga gtt tga tcc tgg ctc ag 1400 [4]


135

DBI acc tat ctc tag gtg tag cgc a Set 8 Ldel-7 aca gat gga tgg aga gca ga 450 [13] Lac-2 cct ctt cgc tcg ccg cta ct Set 9 Laci-1 tgc aaa gtg gta gcg taa gc 210 [13] 23-10C cct ttc cct cac ggt act g Set 10 Lfer 3 act aac ttg act gat cta cga 192 [13] Lfer4 ttc act gct caa gta atc atc Set 11

Lpla2 cct gaa ctg aga gaa ttt ga

Lpla3 att cat agt cta agt tgg agg t 248 [13]

Set 12 Lu1 att gta gag cga ccg aga ag Lac2 cct ctt cgc tcg ccg cta ct

300 [13]

Set 13 Lu3 aaa ccg aga aac acc cgc gtt Lac2 cct ctt cgc tcg ccg cta ct

350 [13]

Set 14 Lu5 cta gcg ggt gcg act ttg tt Lac 2 cct ctt cgc tcg ccg cta ct

400 [13]

Set 15 Lu5 cta gcg ggt gcg act ttg tt RhaII gcg atg ccg aat ttc tat tat t

113 [13]


136

References

1. Dellaglio, F. and Felis, G.E. (2005). Taxonomy of lactobacilli and bifidobacteria. In: Tannock GW, editor. PROBIOTICS AND PREBIOTICS: SCIENTIFIC ASPECTS. UK : Academic Press; p. 25-49.

2. Giraffa, G., Rossetti, L., Neviani, E. (2000). Molecular diversity within Lactobacillus helveticus as revealed by genotypic characterization. Appl. Environ. Microbio, 66: 1259-1265.

3. Nakagawa, T., Shimada, M., Mukai, H., Asada, K. Kato, I, Fujino, K. and Sato, T. (1994). Detection of alcohol-tolerant hiochi bacteria by PCR. Appl. Environ. Microbiol., 60: 637–640

4. Drake, M., Small, C.L., Spence, K.D. and Swanson, B.G. (1996). Rapid detection and identification of Lactobacillus sp. in dairy products by using the polymerase chain reaction. Journal of Food Protection, 59: 1031-1036

5. Quere, F., Deschamps, A. and Urdaci, M.C. (1997). DNA probe and PCR specific reaction for Lactobacillus plantarum. Journal of Applied Microbiology, 82: 783-790.

6. Andrighetto, C., De Dea, P., Lombardi, P., Neviani, E,, Rossetti, L. and Giraffa, G. (1998). Molecular identification and cluster anlaysis of homofermentative thermophilic lactobacilli isolated from dairy products. Research Microbiology, 149: 631-643.

7. Berthier, F., Ehrlich, S, D. (1999). Genetic diversity within Lactobacillus sakei and Lactobacillus curvatus and design of PCR primers for its detection using randomly amplified polymorphic DNA. Int. J. Syst. Bacteriol, 49: 997–1007.

8. Ward, L.J. and Timmins, M.J. (1999). Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction. Letters Apll. Microbiology, 29, 90-92

9. Torriani, S., Felis, G.E. and Dellaglio, F. (1999). Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Applied and Environmental Microbiology, 67, 3450-3454.

10. Walter, J., Hertel, C., Tannock, G.W., Lis, C.M., Munro, K. and Hammes, W.P. (2001). Detection of Lactobacillus, Pediococcus, Leuconostoc and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel

electrophoresis. Appl. Environ. Microbiol. 67, 2578–2585.

11. Yost, C.K. and Nattress, F.M. (2000). The use of multiplex PCR reactions to characterize the populations of lactic acid bacteria associated with meat spoilage. Letters in Applied Microbiology, 31, 129-133.

12. Muller, M.R., Ehrmann, M.A. and Vogel, R.F. (2000). Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation. Appl. Environ. Microbiol., 66, 2113-2116.

13. Song, Y., Kato, N., Liu, C., Matsumiya, Y., Kato, H. and Watanabe, K. (2000) Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA. FEMS Microbiology Letters, 187, 167-173.

14. Chagnaud, P., Machinis, K., Coutte,L.A., Marecat, A. and Mercenier, A. (2001). Rapid PCR based procedure to identify lactic acid bacteria: application to six common bacterial species. J. Microbiol. Mehods, 44: 139-148.

15. Berthier, F., Beuvier, E., Dasen, A. and Grappin, R. (2001). Origin and diversity of mesophilic lactobacilli in Comte cheese as revealed by PCR with repetitive and species specific primers. Int. Dairy and Animal husbandry, 293-305.

16. Heilig, H.G.H.J., Zoetendal, E.G., Vaughan, E.E., Marteau, P., Akkermans, A.D.L. and de Vos, W.M. (2002). Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA. Appl. Environ. Microbiol., 68 :114–123.

17. Dubernet, S., Desmasures, N. and Gueguen, M. (2002). A PCR based method for identification of lactobacilli at the genus level. FEMS Microbiology Letters, 214: 271-275

18. Couret, V., Dubernet, S., Bernadeau, M. Guegen, M. and Vernoux, P. (2001). Isolation, characterization and identification of lactobacilli focusing mainly on cheeses and other dairy products. Le Lait , 83: 269-306.

19. Ikonomopoulus, J., Kokotas, S., Gazouli, M., Zavras, A., Stoitsiou, M., Gorgoulis, V. (2003). Molecular diagnostics of leishmaniosis in dogs. Comparivite application of traditional


137

diagnostic methods and the proposed assay on clinical samples. Vet. Parasitology , 2: 99-113.

20. De Man, J.C., Rogosa, M. and Sharpe, M.E. (1960). Medium for the cultivation of lactobacilli. Journal of Applied Bacteriology, 23: 193 - 198.

21. Terzaghi, B.E. and Sandine, V.E. (1975). Improved medium for lactic streptococci and their bacteriophages. Applied Microbiology, 29, 807 - 813.

22. Official methods of analysis of the Association of official analytical chemists. (1990). 15th ed. Washington DC : The Association, 2 vols.

23. Hensyl, W. R. (Ed.). (1994). Bergey’s manual of systematic bacteriology (9th ed.). Baltimore: Williams & Wilkins.

24. Pospiech, A. and Neumann, B. (1995). A versatile quick preparation of genomic DNA from Gram positive bacteria. TIG , 11, 217-218

25. Sambrook, J., Fritsch, E.F and Maniatis, T. (1989). Molecular cloning: A laboratory manual, 2nd Edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

26. Rochelle, P.A., Fry, J.C., Parkes, J. and Weightman, A.J. (1992). DNA extraction for 16S

rRNA gene analysis to determine genetic diversity deep sediment communites. FEMS Microbiological Letters, 100, 59 - -65.

27. Klijn, N., Weerkamp, A.H. and De Vos, W.M. (1991). Identification of mesophilic lactic acid bacteria by using polymerase chain reaction amplified variable regions of 16S rRNA and specific DNA probes. Appl. Environ. Microbiol., 37, 3390-3393.

28. Weiss, A., Lettner, H.P., Mayer, H.K. and Kneifel, W. (2005). Molecular methods for the identification of potentially probiotic Lactobacillus reuteri strains. Food Technology and Biotechnology, 43 (3), 295-300.

29. De Vecchi, E. and Drago, L. (2006). Lactobacillus sporogenes or Bacillus coagulans : Misidentification Or Mislabelling? International Journal of Probiotics and Prebiotics, Vol. 1, 3-10

30. Drisko, J., Bischoff, B., Giles, C., Adelson, M., Rao, C.V. and McCallum, R. (2005). Evaluation of five probiotic products for label claims by DNA extraction and polymerase chain reaction analysis. Dig Dis Sci., 50(6), 1113-1117.

comparison of specificity of different primer pairs for ...web.firat.edu.tr/ijst/3-2/(1) senan...

Documents