Journal of Microbiology

Journal Article

Performance of nested multiplex PCR assay targeting MTP40 and IS6110 gene sequences for the diagnosis of tubercular lymphadenitis: Pallavi Sinha , Pradyot Prakash , Shashikant C.U. Patne , Shampa Anupurba , Sweety Gupta , G. N. Srivastava; J. Microbiol. 2017;55(1):63-67. Published online December 30, 2016; DOI: https://doi.org/10.1007/s12275-017-6127-y

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The conventional methods for diagnosis of tubercular lymphadenitis (TBLN) such as - fine needle aspiration cytology, Ziehl-Neelsen staining and culture have limitations of low sensitivity and/or specificity. So, it becomes essential to develop a rapid, sensitive, and specific method for an early diagnosis of TBLN. Therefore, the present study was conducted to evaluate nested multiplex polymerase chain reaction (nMPCR) targeting MTP40 and IS6110 gene sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex, respectively in 48 successive patients of TBLN and 20 random patients with non-tubercular lymph node lesions. Out of the 48 cases of TBLN, 14 (29.2%) were found to be positive by Ziehl-Neelsen staining, 15 (31.2%) were positive by culture and 43 (89.6%) cases were positive after first round of PCR while 48 (100%) cases were positive by nMPCR assay. The sensitivity and specificity of nMPCR was found to be 100% for the diagnosis of TBLN. The results thus obtained indicate that nMPCR assay is a highly sensitive and specific tool for the diagnosis of TBLN.

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Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection
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Research Support, Non-U.S. Gov'ts

NOTE] Isolation and Characterization of Histamine-Producing Bacteria from Fermented Fish Products: Jin Seok Moon , So-Young Kim , Kyung-Ju Cho , Seung-Joon Yang , Gun-Mook Yoon , Hyun-Ju Eom , Nam Soo Han; J. Microbiol. 2013;51(6):881-885. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3333-0

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Abstract

Histamine is mainly produced by microorganisms that are found in fermented foods, and is frequently involved in food poisoning. Two histamine-producing bacteria were isolated from fermented fish products, anchovy sauce, and sand lance sauce by using a histidine decarboxylating medium. The species were identified as Bacillus licheniformis A7 and B. coagulans SL5. Multiplex PCR analysis showed the presence of the conserved histidine decarboxylase (hdc) gene in the chromosome of these bacteria. B. licheniformis A7 and B. coagulans SL5 produced the maximum amount of histamine (22.3±3.5 and 15.1±1.5 mg/L, respectively). As such, they were determined to be potential histamine-producing bacteria among the tested cultures.

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Histamine-degrading halophilic bacteria from traditional fish sauce: Characterization of Virgibacillus campisalis TT8.5 for histamine reduction
Thi Thu Hang Tran, Thi Phuong Anh Nguyen, Thi Diu Pham, Thi Hong Nguyen, Thi Lam Doan Nguyen, Thi Thanh Thuy Nguyen, Thi Lan Huong Tran, Trung Khoa Giang, Thi Thu Hien Bui, Bien-Cuong Do, Tien-Thanh Nguyen, Dietmar Haltrich, Hoang Anh Nguyen
Journal of Biotechnology.2023; 366: 46. CrossRef
Detection, Identification, and Inactivation of Histamine-forming Bacteria in Seafood: A Mini-review
Daniel Lance Nevado, Sophia Delos Santos, Gelian Bastian, Jimson Deyta, El-jay Managuelod, Jamil Allen Fortaleza, Rener De Jesus
Journal of Food Protection.2023; 86(3): 100049. CrossRef
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Characterization of Tryptamine-Producing Bacteria Isolated from Commercial Salted and Fermented Sand Lance Ammodytes personatus Sauces
In-Seon Um, Tae-Ok Kim, Hee-Dai Kim, Kwon-Sam Park
Korean Journal of Fisheries and Aquatic Sciences.2016; 49(6): 792. CrossRef
Relationship between chemical characteristics and bacterial community of a Korean salted-fermented anchovy sauce, Myeolchi-Aekjeot
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Isolation and Characterization of Putrescine-producing Bacteria in Commercially Available Sauces Made from Salted and Fermented Sand Lance Ammodytes personatus
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Korean Journal of Fisheries and Aquatic Sciences.2016; 49(5): 573. CrossRef

Prevalence of Amino Acid Changes in the yvqF, vraSR, graSR, and tcaRAB Genes from Vancomycin Intermediate Resistant Staphylococcus aureus: Jae Il Yoo , Jung Wook Kim , Gi Su Kang , Hwa Su Kim , Jung Sik Yoo , Yeong Seon Lee; J. Microbiol. 2013;51(2):160-165. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-3088-7

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Abstract: Vancomycin intermediate Staphylococcus aureus (VISA) strains are increasingly prevalent in the hospital setting, and are of major concern in the treatment of methicillin-resistant S. aureus infections. Multiple mutations in vancomycinsusceptible S. aureus (VSSA) strains likely led to the emergence of VISA, and point mutations in the agr, orf1, yvqF, vraSR, graSR, and tcaRAB genes of VISA strains have been shown to contribute to glycopeptide resistance. Therefore, we investigated point mutations in these genes from 87 VISA and 27 VSSA clinical strains isolated from Korean hospitals. All strains were assigned an agr type (I, II, or III) on the basis of multiplex PCR, with the majority of VISA strains belonging to agr groups I and II. Sequencing revealed amino acid changes in vraS from VISA strains which were not present in the VSSA strains. The E59D substitution in the vraR gene occurred in 36.3% of VSSA/agrI and 92.7% of VISA/agrI strains, suggesting that this mutation associated with emergence of VISA/agrI strains. VISA strains were classified into 31 mutation patterns according to mutations in the yvqF, vraSR, graSR, and tcaRAB genes. In addition, the mutation patterns were correlated with agr and sequence type (ST). The most prevalent pattern included agr type I (ST 72) strains with E59D (vraR), L26F and T224I (graS), D148Q (graR), and L218P, R283H and G312D (tcaA) amino acid substitutions. The minimum inhibitory concentration (MIC) range of mutation pattern 5 toward oxacillin and imipenem was much lower than that of patterns 6 and 24. These results improve our understanding of emergence of VISA strains.

Detection of Representative Enteropathogenic Bacteria, Vibrio spp., Pathogenic Escherichia coli, Salmonella spp., Shigella spp., and Yersinia enterocolitica, Using a Virulence Factor Gene-Based Oligonucleotide Microarray§: Dong-Hun Kim , Bok-Kwon Lee , Yong-Dae Kim , Sung-Keun Rhee , Young-Chang Kim; J. Microbiol. 2010;48(5):682-688. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0119-5

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Abstract: Rapid identification of enteropathogenic bacteria in stool samples is critical for clinical diagnosis and antimicrobial therapy. In this study, we describe the development of an approach that couples multiplex PCR with hybridization to a DNA microarray, to allow the simultaneous detection of the 10 pathogens. The microarray was synthesized with 20-mer oligonucleotide probes that were designed to be specific for virulencefactor genes of each strain. The detection limit for genomic DNA from a single strain was approximately 10 fg. In the presence of heterogeneous non-target DNA, the detection sensitivity of the array decreased to approximately 100 fg. We did not observe any non-specific hybridization. In addition, we successfully used this oligonucleotide-based DNA microarray to identify the causative agents in clinical stool samples from patients with food-borne enteritis.

Isolation and Characterization of Biogenic Amine-Producing Bacteria in Fermented Soybean Pastes: Jin Seok Moon , Seung Kee Cho , Hwa Young Choi , Ji Eun Kim , So-Young Kim , Kyung-Ju Cho , Nam Soo Han; J. Microbiol. 2010;48(2):257-261. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-0040-y

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Abstract: Biogenic amines (BAs) are produced primarily by microorganisms found in fermented foods and are often implicated in food poisoning. BA-producing bacteria found in fermented soybean pastes were isolated and characterized using a decarboxylating medium and multiplex PCR analysis. Two BA-producing bacteria were isolated from traditional soybean pastes: one was a histamine-producing Clostridium strain, and the other was a tyramine-producing Pseudomonas strain. The Clostridium strain was determined to be a potent histamine producer among the cultures tested. Synthesis of tyramine by Pseudomonas sp. T1 was observed for the first time in this study.

Rapid One Step Detection of Pathogenic Bacteria in Urine with Sexually Transmitted Disease (STD) and Prostatitis Patient by Multiplex PCR Assay (mPCR): Sang Rok Lee , Ji Min Chung , Young Gon Kim; J. Microbiol. 2007;45(5):453-459.; DOI: https://doi.org/2590 [pii]

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Abstract: We developed a multiplex PCR (mPCR) assay to simultaneously detect Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Ureaplasma urealyticum, Corynebacterium spp. and seudomona aeruginosa. This method employs a single tube and multiple specific primers which yield 200, 281, 346, 423, 542, and 1,427 bp PCR products, respectively. All the PCR products were easily detected by agarose gel electrophoresis and were sequenced to confirm the specificity of the reactions. To test this method, DNA extracted from urine samples was collected from 96 sexually transmitted disease or prostatitis patients at a local hospital clinical center, and were subjected to the mPCR assay. The resulting amplicons were cloned and sequenced to exactly match the sequences of known pathogenic isolates. N. gonorrhoeae and Corynebacterium spp. were the most frequently observed pathogens found in the STDs and prostatitis patients, respectively. Unexpectedly, P. aeruginosa was also detected in some of the STD and prostatitis samples. More than one pathogen species was found in 10% and 80.7% of STD and prostatitis samples, respectively, indicating that STD and prostatitis patients may have other undiagnosed and associates. The sensitivity of the assay was determined by sing purified DNA from six pathogenic laboratory strains and revealed that this technique could detect pathogenic DNA at concentrations ranging from 0.018 to 1.899 pg/μl. Moreover, the specificities of this assay were found to be highly efficient. Thus, this mPCR assay may be useful for the rapid diagnosis of causative infectious STDs and prostatitis. useful for the infectious STDs and prostatitis.

Validation Study

Differentiation of Lymphocystis Disease Virus Genotype by Multiplex PCR: Shin-Ichi Kitamura , Sung-Ju Jung , Myung-Joo Oh; J. Microbiol. 2006;44(2):248-253.; DOI: https://doi.org/2358 [pii]

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Abstract: Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease. The viruses have been divided into three genotypes (genotype I for LCDV-1, II for Japanese flounder isolates, and III for rockfish isolates) on the basis of major capsid protein (MCP) gene sequences. In this study, we developed a multiplex PCR primer set in order to distinguish these genotypes. We also analyzed the MCP gene of a new LCDV isolate from the sea bass (SB98Yosu). Comparison of sequence identities between SB98Yosu and eight Japanese flounder isolates, revealed identity of more than 90.1% at nucleotide level and 96.5% at deduced amino acid level, respectively. Phylogenetic analyses based on the MCP gene showed that SB98Yosu belongs to genotype II, along with Japanese flounder isolates. Multiplex PCR based on the MCP gene allowed us to identify these genotypes in a simple and rapid manner, even in a sample that contained two genotypes, in this case genotypes II and III.

Research Support, Non-U.S. Gov't

Detection of Escherichia coli O157:H7, Salmonella spp.,Staphylococcus aureus and Listeria monocytogenes in Kimchi by Multiplex Polymerase Chain Reaction (mPCR): Yeon Sun Park , Sang Rok Lee , Young Gon Kim; J. Microbiol. 2006;44(1):92-97.; DOI: https://doi.org/2331 [pii]

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Abstract: We developed an mPCR assay for the simultaneous detection, in one tube, of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes using species-specific primers. The mPCR employed the E. coli O157:H7 specific primer Stx2A, Salmonella spp. specific primer Its, S. aureus specific primer Cap8A-B and L. monocytogenes specific primer Hly. Amplification with these primers produced products of 553, 312, 405 and 210 bp, respectively. All PCR products were easily detected by agarose gel electrophoresis, and the sequences of the specific amplicons assessed. Potential pathogenic bacteria, in laboratory-prepared and four commercially available kimchi products, were using this mPCR assay, and the amplicons cloned and sequenced. The results correlated exactly with sequences derived for amplicons obtained during preliminry tests with known organisms. The sensitivity of the assay was determined for the purified pathogen DNAs from four strains. The mPCR detected pathogen DNA at concentrations ranging from approximately 0.45 to 0.05 pM/μl. Thus, this mPCR assay may allow for the rapid, reliable and cost-effective identification of four potentially pathogens present in the mixed bacterial communities of commercially available kimchi.

Multiplex Polymerase Chain Reaction Assay for Simultaneous Detection of Candida albicans and Candida dubliniensis: Young-Hee Lim , Do-Hyun Lee; J. Microbiol. 2002;40(2):146-150.

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Abstract: A multiplex polymerase chain reaction (PCR) assay was developed for the identification of two Candida species-albicans and dubliniensis. Three sets of primers were selected from different genomic sequences to specifically amplify a 516 bp fragment within the top2 gene, specific for several species of the genus Candida (CCL primers); a 239 bp fragment within the [alpha]INT1 gene, specific for Candida albicans (CAL primers); and a 175 bp fragment within the ALSD1 gene, specific for Candida dubliniensis (CDL primers). Using the primers in conjunction (multiplex PCR), we were able to detect both C. albicans and C. dubliniensis and to differentiate between them. The detection limit of the PCR assay was approximately 10 cells per milliliter of saline. Thus, this multiplex PCR assay can be applied for differentiation of C. albicans and C. dubliniensis from clinical specimens.

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