Multivariate Optimization of Polymerase Chain Reaction for Microbial Community Analysis

2002 ◽  
Vol 4 (4) ◽  
pp. 423-430 ◽  
Author(s):  
Ingela Dahllöf ◽  
Staffan Kjelleberg
Keyword(s):  
Polymerase Chain Reaction ◽  
Microbial Community ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Multivariate Optimization ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Deconstructing the Polymerase Chain Reaction II: an improved workflow and effects on artifact formation and primer degeneracy

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7121
Author(s):  
Ankur Naqib ◽  
Silvana Poggi ◽  
Stefan J. Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Microbial Community ◽  
Annealing Temperature ◽  
Degenerate Primer ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Polymerase Chain ◽  
Dna Template ◽  
Microbial Dna

Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias. In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

scholarly journals Isolation and Identification of Lactic Acid Bacteria from Natural Whey Cultures of Buffalo and Cow Milk

Foods ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 233
Author(s):  
Rosangela Marasco ◽  
Mariagiovanna Gazzillo ◽  
Nicoletta Campolattano ◽  
Margherita Sacco ◽  
Lidia Muscariello
Keyword(s):  
Polymerase Chain Reaction ◽  
Lactic Acid ◽  
Microbial Community ◽  
Lactic Acid Bacteria ◽  
Microbial Diversity ◽  
Strain Level ◽  
Sequencing Analysis ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Polymerase Chain

In southern Italy, some artisanal farms produce mozzarella and caciocavallo cheeses by using natural whey starter (NWS), whose microbial diversity is responsible for the characteristic flavor and texture of the final product. We studied the microbial community of NWS cultures of cow’s milk (NWSc) for the production of caciocavallo and buffalo’s milk (NWSb) for the production of mozzarella, both from artisanal farms. Bacterial identification at species and strain level was based on an integrative strategy, combining culture-dependent (sequencing of the 16S rDNA, species/subspecies-specific Polymerase Chain Reaction (PCR) and clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and culture-independent (next-generation sequencing analysis, NGS) approaches. Results obtained with both approaches showed the occurrence of five species of lactic acid bacteria in NWSb (Lactococcus lactis subsp. lactis, Lactobacillus fermentum, Streptococcus thermophilus, Lactobacillus delbrueckii, and Lactobacillus helveticus) and five species in NWSc (Lc. lactis subsp. lactis, Enterococcus faecium, and S. thermophilus, Lb. helveticus, and Lb. delbrueckii), with the last two found only by the NGS analysis. Moreover, RAPD profiles, performed on Lc. lactis subsp. lactis different isolates from both NWSs, showed nine strains in NWSb and seven strains in NWSc, showing a microbial diversity also at strain level. Characterization of the microbiota of natural whey starters aims to collect new starter bacteria to use for tracing microbial community during the production of artisanal cheeses, in order to preserve their quality and authenticity, and to select new Lactic Acid Bacteria (LAB) strains for the production of functional foods.

scholarly journals Bacterial community analysis in the rice straw sample before and after composting by PCR-DGGE and cloning method

2020 ◽  
Vol 18 (1) ◽  
pp. 177-186
Author(s):  
Ngo Duc Duy ◽  
Nguyen Hoang Dung ◽  
Hoang Quoc Khanh
Keyword(s):  
Microbial Community ◽  
Rice Straw ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Bacillus Species ◽  
Study Results ◽  
Cloning Method ◽  
Before And After ◽  
Polymerase Chain ◽  
Bacterial Groups

Applicability of PCR-DGGE (Polymerase Chain Reaction-Denaturing Gel Gradient Eletrophoresis) technique to analyze microbial community is based on the V3 gene fragments in the rice straw sample (R) and after composting rice straw samples (Rn). Besides clonning method combined with analyzing Rn samples and comparing the results of microbial community analysis with PCR-DGGE method had the same reuslts is the main goal in this study. Results obtained from PCR-DGGE technique of R sample had 5 the V3 gene fragments (R1, R2, R3, R4 and R5) and Rn had 4 the V3 genes fragments (Rn1, Rn2, Rn3 and Rn4). Comparison of bacterial groups in R and Rn samples showed that bacteria in R samples including Agrobacterium, Clostridium, Bacteroidetes, Thermopolysporasa and Bacillus species, but in the Rn sample after incubation, the remaining bacterial results of 2 main species are Agrobacterium and Clostridium. Using Clonning method of Rn sample also gave 4 positions and gene fragment sizes corresponding to the positions of Rn1, Rn2, Rn3 and Rn4 from representatives of about 30 samples collected from the Clonning products. Based on the comparison of the similarity among the sequence of V3 gene fragments in PCR-DGGE and Clonning with reference to the data base in NCBI gene bank the results that Rn1 and Rn4 genes are similar to Agrobacterium species, about 96%, Rn2 and Rn3 are similar to Clostridium about 99%. In summary the results of microbial community analysis in the R sample show that the diversity of bacteria in the R sample is larger than in the Rn sample. Summarize the results of microbial community analysis in the R sample show the diversity of bacteria but less stable than the Rn sample. In addition, cloning and PCR-DGGE produced similar results on the sample Rn.

scholarly journals Deconstructing the Polymerase Chain Reaction II: An improved workflow and effects on artifact formation and primer degeneracy

2019 ◽  
Author(s):  
Ankur Naqib ◽  
Silvana Poggi ◽  
Stefan J Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Microbial Community ◽  
Annealing Temperature ◽  
Degenerate Primer ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Polymerase Chain ◽  
Dna Template ◽  
Microbial Dna

Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias (Green et al. 2015). In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

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