Journal of Microbiology

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Variations in 16S rRNA-based Microbiome Profiling between Pyrosequencing Runs and between Pyrosequencing Facilities: Minseok Kim , Zhongtang Yu; J. Microbiol. 2014;52(5):355-365. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3443-3

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Pyrosequencing of 16S rRNA gene amplicons on the 454 FLX Titanium platform has been widely used to analyze microbiomes in various environments. However, different results may stem from variations among sequencing runs or among sequencing facilities. This study aimed to evaluate these variations between different pyrosequencing runs by sequencing 16S rRNA gene amplicon libraries generated from three sets of rumen samples twice each on the 454 FLX Titanium system at two independent sequencing facilities. Similar relative abundances were found for predominant taxa represented by large numbers of sequence reads but not for minor taxa represented by small numbers of sequence reads. The two sequencing facilities revealed different bac-terial profiles with respect to both predominant taxa and minor taxa, including the most predominant genus Prevo-tella, the family Lachnospiraceae, and the phylum Proteo-bacteria. Differences in primers used to generate amplicon libraries may be a major source of variations in microbiome profiling. Because different primers and regions of 16S rRNA genes are often used by different researchers, significant variations likely exist among studies. Quantitative interpre-tation for relative abundance of taxa, especially minor taxa, from prevalence of sequence reads and comparisons of re-sults from different studies should be done with caution.

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Development of SCAR Primers Based on a Repetitive DNA Fingerprint for Escherichia coli Detection: Aphidech Sangdee , Sitakan Natphosuk , Adunwit Srisathan , Kusavadee Sangdee; J. Microbiol. 2013;51(1):31-35. Published online March 2, 2013; DOI: https://doi.org/10.1007/s12275-013-2244-4

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Abstract: The present study aimed to use enterobacterial repetitive intergenic consensus (ERIC) fingerprints to design SCAR primers for the detection of Escherichia coli. The E. coli strains were isolated from various water sources. The primary presumptive identification of E. coli was achieved using MacConkey agar. Nineteen isolates were selected and confirmed to be E. coli strains based on seven biochemical characteristics. ERIC-PCR with ERIC 1R and ERIC 2 primers were used to generate DNA fingerprints. ERIC-PCR DNA profiles showed variant DNA profiles among the tested E. coli strains and distinguished all E. coli strains from the other tested bacterial strains. A 350 bp band that predominated in five E. coli strains was used for the development of the species-specific SCAR primers EC-F1 and EC-R1. The primers showed good specificity for E. coli, with the exception of a single false positive reaction with Sh. flexneri DMST 4423. The primers were able to detect 50 pg and 100 CFU/ml of genomic DNA and cells of E. coli, respectively.

NOTE] Development of Porphyromonas gingivalis-Specific Quantitative Real-Time PCR Primers Based on the Nucleotide Sequence of rpoB: Soon-Nang Park , Jae-Yoon Park , Joong-Ki Kook; J. Microbiol. 2011;49(2):315-319. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-1028-y

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Abstract: Species-specific quantitative real-time PCR (qPCR) primers were developed for the detection of Porphyromonas gingivalis. These primers, Pg-F/Pg-R, were designed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). Species-specific amplicons were obtained from the tested P. gingivalis strains but not in any of the other strains (46 strains of 46 species). The qPCR primers could detect as little as 4 fg of P. gingivalis chromosomal DNA. These findings suggest that these qPCR primers are suitable for applications in epidemiological studies.

Evaluation of the Sensitivity and Specificity of Primer Pairs and the Efficiency of RNA Extraction Procedures to Improve Noroviral Detection from Oysters by Nested Reverse Transcription-Polymerase Chain Reaction: Cheonghoon Lee , Sooryun Cheong , Hee-Jung Lee , Miye Kwon , Ilnam Kang , Eun-Gyoung Oh , Hong-Sik Yu , Soon-Bum Shin , Sang-Jong Kim; J. Microbiol. 2010;48(5):586-593. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0047-4

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Abstract: Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoVpolluted sea areas are considered as the significant vectors of viral transmission. To improve NoV detection from oyster using nested reverse transcription-polymerase chain reaction (RT-PCR), we evaluated the sensitivity and specificity of previously published primer pairs and the efficiency of different RNA extraction procedures. Among the primer pairs used for RT-PCR, the sensitivity of GIF1/GIR1-GIF2/GIR1 and GIIF1/GIIR1-GIIF2/GIIR1 was higher than that of other primer pairs used in nested RT-PCR for the detection of NoV genogroup I (NoV GI) and NoV GII from both NoV-positive stool suspension and NoVseeded oyster concentrates, respectively; the resulting products showed neither unspecific bands in the positive samples nor false-positive bands in the negative controls. The extraction of NoV RNA from oyster samples using a QIAamp? Viral RNA Mini kit with a QIAshredderTM Homogenizer pretreatment afforded more efficient recovery (mean recovery for NoV GI and GII, 6.4%) and the procedure was less time consuming (<30 min) than most other RNA extraction procedures. The results of RNA extraction procedure and primer pairs evaluated by nested RT-PCR assay in this study can be useful for monitoring NoV contamination in oysters, which is an indicator of possible public health risks.

Designing Primers from Multiple Sequences Using Matchup Program to Improve Detection of Hepatitis B Virus by Polymerase Chain Reaction: So Young Jang , Mi Suk Kim , Min Seok Park , Keon Myung Lee , Hwan Won Chung , Jongsik Chun , Chan Hee Lee; J. Microbiol. 2010;48(1):111-116. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0282-8

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Abstract: Traditionally primers for PCR detection of viruses have been selected from genomic sequence of single or representative viral strain. However, high mutation rate of viral genomes often results in failure in detecting viruses in clinical and environmental samples. Thus, it seems necessary to consider primers designed from multiple viral sequences in order to improve detection of viral variants. Matchup is a program intended to select universal primers from multiple sequences. We designed using Matchup program primer pairs for HBV detection from 691 full genomic HBV DNA sequences available from NCBI GenBank database. Thousands of primer candidates were initially extracted and these were sequentially filtered down to 5 primer pairs. These primer pairs were tested by PCR using 5 HBV Korean HBsAg(+) patient sera, and eventually one universal primer pair was selected and named MUW (multiple-universal-worldwide). This primer pair, 3 HBV reference primer pairs reported by others and 1 commercial primer pair were compared using 86 HBV HBsAg(+) sera from Korean and Vietnamese patients. The detection rate for MUW primer pair was 72.1%, much greater than those obtained by reference and commercial primers (32.5 to 40.7%). The superiority of MUW primer pair appeared to be correlated with the conserved sequences of the forward primer binding sites and primer quality score. These results suggest that the universal primers designed by the Matchup program from multiple sequences could be useful in detecting viruses from clinical samples.

Development of Strain-specific PCR Primers Based on a DNA Probe Fu12 for the Identification of Fusobacterium nucleatum subsp. nucleatum ATCC 25586^T: Hwa-Sook Kim , Soo Keun Song , So Young Yoo , Dong Chun Jin , Hwan Seon Shin , Chae Kwang Lim , Myung-Soo Kim , Jin-Soo Kim , Son-Jin Choe , Joong-Ki Kook; J. Microbiol. 2005;43(4):331-336.; DOI: https://doi.org/2257 [pii]

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Abstract: The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC 25586^T (F. nucleatum ATCC 25586^T), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC 25586^T. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC 25586^T. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC 25586^T, especially with regard to the determination of the authenticity of the strain.

Identification of Actinobacillus actinomycetemcomitans Using Species-Specific 16S rDNA Primers: Su-Gwan Kim , Soo-Heung Kim , Mi-Kwang Kim , Hwa-Sook Kim , Joong-Ki Kook; J. Microbiol. 2005;43(2):209-212.; DOI: https://doi.org/2159 [pii]

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Abstract: The purpose of this study was to develop species-specific PCR primers for use in the identification and detection of Actinobacillus actinomycetemcomitans. These primers target variable regions of the 16S ribosomal RNA coding gene (rDNA). We assessed the specificity of the primers against 9 A. actinomycetemcomitans strains and 11 strains (3 species) of the Haemophilus genus. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC 33384^T. Our obtained data revealed that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 4 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these PCR primers are incredibly sensitive, and should prove suitable for application in epidemiological studies, as well as the diagnosis and monitoring of periodontal pathogens after treatment for periodontitis.

Genetic Variation of BIV Isolates Characterized by PCR Using Degenerate Primers: Kwon, Oh Sik , Sninsky, John J.; J. Microbiol. 1995;33(3):252-259.

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Abstract: The PCR was employed to detect and characterize the bovine immunodeficiency-like virus (BIV), which is a newly recognized member of the Lentivirinae of the retroviruses. Degenerate primers representing the conserved regions in the pol genes of the Lentivirinae, were used to detect proviral DNA obtained from the bovine embryonic spleen cell cultures infected with BIV. The PCR amplified DNA fragment was molecularly cloned and sequenced. The BIV DNA fragment contained a sequence identical to that reported by Garvey et al. (Garvey et al., 1990. Virology, 175, 391-409). With the degenerate primers, peripheral blood mononuclear cells (PBMCs) of sick cattle and cells cultured with BIV were tested to determine genetic variation of BIV pol conserved sequence. We found the sequence heterogeneity within cultures and most variations occurred at the third base of codons that would not lead to amino acid substitutions. Another change was GAG (Glu) to AAG (Lys) within the BIV isolates. Interestingly, the altered sequence is also found in other lentiviruses such as HIV-2, SIV mac, CAEV and EIAV.

Evaluation of Several Parameters of in situ Polymerase Chain Reaction (ISPCR) to Reduce the Leakage of Amplificants from Cells: Jae Yung Lee , Chung-Kyoon Auh , George W. Jordan; J. Microbiol. 2002;40(1):70-76.

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Abstract: Proviral DNAs from HIV-1-infected CD4+ T cells (Molt/LAV cells) were amplified and detected in infected individual cells using polymerase chain reaction and in situ hybridization. In this in situ PCR, three parameters were considered to achieve effective amplification and retention of amplificants inside the cells by making high molecular weight PCR products intracellularly, forming agarose matrix against the cells, and maintaining the appropriate PCR temperature profile. Over the cycles of amplification, tailed primers with complementary overhanging sequences at their 5 sides manufactured high molecular weight products by using short primary products as a repeating unit. Agarose matrix could prevent the diffusion of the amplificants from the cells. Use of Thermanox coverslip inside the PCR tube offered target cells a similar temperature profile to that of conventional PCR in solution.

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