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2. Advancement in LH-PCR methodology for multiple microbial species detections in fermented foods Presented to : Dr. Aqeela Ashraf Presented by: Zobia Javed ( 001), Sahar Noor (002), Samiya Habib (006),H. Akifa Naeem (007), Muneeba Nahid (008 ) Biology Department, LGU, Lahore 2 Recombinant DNA Technology MPhil Zoology-II Journal: Food Microbiology Impact Factor: 4.155

3. WOODGROVE BANK Contents

4. Introduction Sahar Noor (003) 4

5. Methods for microbial species detections 5 Denaturing and temperature gradient gel electrophoresis (DGGE and TGGE), single-strand conformation polymorphism (SSCP ), terminal-restriction fragment length polymorphism (tRFLP) Length heterogeneity PCR (LH-PCR ) Why LH-PCR? simpler , more widely used, more cost effective and don't require large automated facilities. Previously LH-PCR is being used in the characterization of bacterial communities involved in biofilm, bio-treatment and bioremediation, mostly for environmental problems Also, in human and animal disease and infections identification. Recently, it is confirmed that the LH-PCR could be a promising method to be used in food microbiology analysis, not only for fingerprinting analysis but also for direct species identification through the use of a specific database by, Garofalo et al. (2017a ), Lazzi et al., (2004) and Gatti et al., 2008. The specific database used: the LH-PCR database available on Lactic Acid Bacteria (LAB)

6. LH-PCR (length-heterogeneity PCR) 6 L ength heterogeneity PCR (LH-PCR ), a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms . Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. The sizes of the fragments on the polyacrylamide gel after electrophoresis can be compared against 16S rRNA gene databases to specify microbial groups that may correspond in size to the size of the fragments.

7. Why we need to optimize LH-PCR? 7 A limiting factor which relates to the high percentage of secondary peaks . T he LH-PCR uses amplification of the region of the rRNA gene operon between the small (16S) and large (23S) subunits called the intergenic spacer (IS) region. To detect and identify the Bacteria. T he limitation lies in the unpredictability and random pattern of variations in spacer size. : i) data interpretation analysis; ii) overestimation of the bacterial species present in complex matrices and their relative quantification. In the case of multiple ribosomal operons within a strain, the sizes and sequences of different 16S-23S PCR amplicons may vary considerably in a single bacterial cell. In community profiling this means that a single species may contribute more than one peak or band to the community profile.

8. Objectives Q# Whether LH-PCR can be used for food microbial species detections in fermented foods ? To optimize LH-PCR to overcome the problem of high percentage of secondary peaks . 8

9. What they have done? Tried to identify the optimal PCR conditions which are able to maintain the bacteria discrimination but at the same time increasing the accuracy of the PCR analysis . T he new PCR conditions were tested mainly on products fermented by LAB such as Crescenza, raw milk cheese and olives . T he robustness of the new PCR conditions were also evaluated by analyzing products obtained with different fermentation processes such as: alcoholic and/or lactic acid fermentation (kefir, sourdough, fermented sausage) and acetic fermentation (vinegar ). The strains previously considered by Lazzi et al., (2004) and Gatti et al., 2008 were also analyzed with the new PCR condition with the aim to produce a wider integrated database. 9

10. Materials 10 Samiya Habib ( 006

11. Materials Chemicals: Chemicals Quantity Purpose Chemicals Quantity Purpose Ringer Solution 90 ml Create an isotonic solution relative to the body fluid glucose, yeast extract, calcium carbonate, AE-medium For isolation of acetic bacteria de Man, Rogosa and Sharpe agar For isolation of lacto-bacilli glycerol 1 ml use as cryoprotective agent pediococci selective medium use for pediococci isolation agarose gel agarose gel 1% M17 For lactococci,enterococci, spteptococci isolation 1X Taq Buffer 19ul  agarose electrophoresis Manitol Salt Agar Use for streptococci isolation de-ionized form amide 12ul Useful for preparing hybridization buffer 11

12. Materials Chemicals: Chemicals Quantity Purpose primers 46F, 536R, forward primer, 63F, reverse primers 355R, 0.2 mM Use for initiation of DNA synthesis MgCl2 1.5 mM essential cofactor that enhances the activity of Taq DNA polymerase dNTPs 0.2 mM Use in PCR expand the growing DNA strand Taq DNA polymerase 1U Use in PCR role in synthesizing & amplifying new strand of DNA phosphoramidite dye 6-FAM fluorescein dye designed for detection and analysis of labeled PCR products 12

13. Materials Apparatuses: Apparatus Purpose Apparatus Purpose Stomacher To wash and extract intact microbes in solution ABI Prism 310 Genetic Analyser Perform comparative sequencing, linkage analysis centrifuge For extraction of DNA pellet Gene Mapper ver. 4.0 software The size LH-PCR fragment was estimated QIAamp DNA Stool Mini Kit Extracts DNA from complex bacterial ecosystem Electrophoresis tank Evaluated the presence & quality of extracted DNA Thermal Cycler PCR apparatus SV Gel & PCR-clean up system Extract and purify DNA fragment of 100bp to 10kp from standard agarose gel 13

14. 14 Methods H. Akifa Naeem ( 007

15. Methods Steps: 15

16. Sampling of fermented food and bacteria strains isolation The samples were diluted tenfold using Ringer solution and plated onto selective media to isolate the following microbial groups Isolated strains were also cultivated overnight at an optimal temperature, i.e. 42 C for thermophilic LAB and 30-37 C for all the others. The isolated strains were stored as frozen stock at 80 C with 1 ml of glycerol as cryoprotective agent . Chemicals Working Apparatus Chemicals Working Ringer Solution Samples of 10 g or 10 ml were homogenized solution using a Stomacher for 5 min M17 at 30 C for 48 h for lactococci, at 37 C for 48 h enterococci and at 42 C for 48 h for streptococci. de Man, Rogosa and Sharpe agar At 35 C for 48 hrs. for lactobacilli Manitol Salt Agar at 37 C for 48 h for staphylococci pediococci selective medium (PSM) at 37 C for 48 hrs. for pediococci glucose, yeast extract, calcium carbonate, AE-medium at 30 C for 27days for acetic bacteria. 16 1 st Step

17. DNA extraction from fermented food samples and bacterial strains 17 2 nd Step The presence and quality of the extracted DNA was evaluated using 1% (w/v) agarose gel-electrophoresis.

18. Bacteria strains identification 18 3 rd Step

19. LH-PCR amplification – annealing temperature evaluation 19 Total DNA extracted from fermented food samples and from bacterial strains were analyzed following the LH-PCR amplification To identify the most specific annealing temperatures for the fermented food samples and bacterial strains different temperature ranging from 49C to 65 C were evaluated. Domain A of the variable regions of the 16S rRNA gene from extracted DNA was amplified. The forward primer, 63F (50-CAGGCCTAACACATGCAAGTC-30) was 5’ end labeled with the phosphoramidite dye 6-FAM and the reverse primers used were 355R (5’-GCT GCC TCC CGT AGG AGT-3’). In each PCR amplification, 1 ml of extracted DNA was added to 19 ml of the amplification mixture, resulting in a final concentration of 1X Taq Buffer, 1.5 mM of MgCl2, 0.2 mM of dNTPs, 0.2 mM of each primer, and 1U of Taq DNA polymerase (Promega), in a final reaction volume of 20 ml. PCR conditions were as follows: an initial denaturation at 95 C for 5 min, 25 cycles of denaturation at 95 C for 30s different annealing temperature were used (49, 51, 53, 56, 59, 63 C and 65 C) for 30s elongation at 72 C for 1 min 30s, and a final extension step at 72 C for 7 min. PCR product amplified were diluted 15 time fold for subsequent fragment analysis as described below . 4 th Step

20. Results 20 Muniba Naheed (008) Zobia Javed (001) Muniba Naheed (21-24) Zobia Javed (25-26)

21. Efficiency of primer annealing 21 Efficiency of primer annealing is a very important factor for the success and stringency of PCR . Different culture media were used to isolate bacterial strains from these food samples. 41 strains from cheese,42 strains from vinegar, 57 strains from sourdough were obtained. These isolates were analyzed by LH-PCR using 49 C as annealing temperature and were then grouped according to the amplicons profile. Amplicons profile are 21 from cheese, 9 from vinegar, 28 from sourdough. It was found that strains of same specie showed different amplicon profiles in LH-PCR. From the sourdough, 28 different amplicon profiles were Found. Only 3 different species were identified by 16S gene sequencing: Strains isolated from raw-milk cheeses among 21 different profiles. 4 species were identified by 16S gene sequencing.

22. Efficiency of primer annealing 22 Advantages:

23. PCR reactions using different annealing temperatures 23 49C - 65 C was used to identify the most specific one for bacteria discrimination, to reduce the unspecific amplification and also the secondary peaks and for high amplification efficiency. By testing different temperature it was found in Fig. 1, that the Increase in the annealing temperature determined a reduction in the incidence of the secondary peaks. The chromatogram obtained shows that for L. paralimentarius 4486 the presence of three main peaks occurred at lower annealing temperatures 49-59C . But by increasing the temperature to 63 C caused the main peak of 337 bp to be maintained , but the secondary peaks were reduced/removed. Like wise For Weissela minor 7 peaks at 49 C and one major peak of 346 bp at 63 C . With Leuconostoc citreum six peaks at 49 C and one main peak of 316 bp at 63 C. The same results were obtained for all strains .

24. PCR reactions using different annealing temperatures 24 It proved LH-PCR analysis of bacteria strains isolated from cheese the optimal annealing temperature is 65 C and for other strains isolated temperature was 63C. I t means reduction/elimination of the secondary and unspecific peaks occurred at higher annealing temperature .

25. Effects of annealing temperature on PCR efficiency- fermented food samples 25 The effect of the annealing temperature was also evaluated on the fermented food samples using 2 samples of raw cow's milk cheese, 2 samples of vinegar and 2 samples of sourdough. The chromatogram shows that the increase in the annealing temperature leads to improve the distinctiveness of the different peaks. But the annealing temperature of 63-65C , used in the previous experiment with bacteria strains, was found to be too restrictive towards the amplification profiles of food matrices. The optimal temperature was found to be 59 C for fermented food samples as it was able to maintain the total variability of the species found within the matrices but reducing the background related to unspecific products. This may occur because in food matrices the micro flora is extremely complex (with bacterial populations with high and low concentrations), so high annealing temperatures can be too stringent and fail to identify species that are in low concentration. Zobia Javed

26. Effects of annealing temperature on PCR efficiency- fermented food samples 26 The optimal annealing temperature found (59 C) was tested on another four fermented foods of both animal and vegetable origin, in order to evaluate the robustness of the parameter identified. It is evident that the peaks in the chromatograms are well distinguished, with a good fluorescence signal, and comparing them with the database and also with the sequenced strains obtained, is also possible to identify most of the species present in the matrices. In order to qualify the possibility to evaluate the LH-PCR products without capillary electrophoresis equipment, an experiment using high-resolution agarose gel was performed. The LH-PCRs of two food matrixes amplified at 59 C (fermented olives and salami) and their isolated bacteria strains amplified at 63 C , were evaluated with 3% high-resolution agarose gel. The gel confirms the correspondence of the species-specific bands in the relative food matrixes and also the bands intensity are correlated with their abundance in the food matrixes as it is showed also by the capillary electrophoresis chromatogram.

27. Conclusion 27 Zobia Javed ( 001

28. Conclusion 28 The approach presented in this work allowed the modification of the previous LH-PCR protocol with the identification of the optimal annealing temperatures that enables an increasing in the amplification efficiency and therefore also in the bacteria species discrimination. In particular, the optimal annealing temperature suggested for fermented food samples is 59 C and the optimal annealing temperature for bacterial strains varied between 63 or 65 C , depending on the food source of the strains. The modification in the level of accuracy of the LH-PCR technique could also allow an improvement in the relative species quantification by considering the peak area evaluation.

29. Citations 29 Sahar noor (003)

30. Image SLide Published : September 2018 Citations: Bertani , G., Sardaro, M. L. S., Neviani, E., & Lazzi, C. ( 2019 ). AFLP protocol comparison for microbial diversity fingerprinting .  Journal of applied genetics ,  60 (2), 217-223 . (April-2019) Journal of Applied Genetics: Impact Factor: 2.027 Menezes, L. A. A., Sardaro, M. S., Duarte, R. T. D., Mazzon, R. R., Neviani, E., Gatti, M., & Lindner, J. D. D. ( 2020 ). Sourdough bacterial dynamics revealed by metagenomic analysis in Brazil.  Food microbiology ,  85 , 103302. (Feb-2020) Journal : Food Microbiology: Impact Factor: 4.155 30 Advancement in LH-PCR methodology for multiple microbial species detections in fermented foods

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32. 32 Thank You