Comparative lactic acid bacteria (LAB) profiles during dadih fermentation with spontaneous and back-slopping methods, as identified by terminal-restriction fragment length polymorphism (T-RFLP)

The diversity of lactic acid bacteria (LAB) present during the manufacture of traditional fermented buffalo milk from West Sumatra, known as dadih, was studied via a culture-independent approach using terminal-restriction fragment length polymorphism (T-RFLP) to compare the dynamic diversity in back-slopping and spontaneous fermentation methods. Total LAB and pH were measured in freshly prepared buffalo milk and in \textit{dadih} fermented for 24 and 48 hours. The results indicated significant differences between the fermentation methods, with higher total LAB, and greater phylotype richness and relative abundance being identified in the back-slopping method. Terminal fragment lengths (TRFs) of 68 and 310 bp were common to both techniques, similar to those of Lactobacillus fermentum, Fructobacillus pseudoficulneus, Leuconostoc citreum, Leuconostoc kimchii, and Leuconostoc sp. The changes in phylotype number (species number) and relative abundances of LAB communities identified are expected to produce data needed to formulate the best fermentation process for dadih manufacturing. A 24-hour back-slopping fermentation method is recommended, as fermentation time of longer than 24 hours reduced viable LAB significantly. Our results also indicated that the T-RFLP technique is not only clearly sensitive enough and adequate for segregating LAB diversity in both fermentation methods, but that it also provides good information regarding the structure of microbial communities and their composition change during the fermentation process.

Evaluation of PCR pncA-restriction fragment length polymorphism and PCR amplification of genomic regions of difference for the identification of M. bovis strains in lymph nodes cultures

Background: A rapid accurate identification of Mycobacterium bovis is essential for surveillance purposes. Objectives: A PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of 3 regions of difference (RD-PCR): RD9, RD4 and RD1 were evaluated for the identification of M. bovis in lymph nodes cultures, in Tunisia, during 2013-2015. Methods: Eighty-two M. tuberculosis complex strains were identified using the biochemical tests, GenoType MTBC assay, PCR pncA-RFLP and RD-PCR. Results: The PCR pncA-RFLP showed that 54 M. bovis strains, identified by GenoType MTBC, had a mutation at position 169 of pncA gene. Twenty-eight strains did not show any mutation at this position 27 M. tuberculosis isolates and one M. caprae. The PCR pncA-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0) and a specificity of 100.0% (95%CI: 87.9- 100.0) for identifying M. bovis. The RD-PCR showed that all M. bovis strains had the RD9 and RD4 deleted but presented RD1. RD-PCR also presented high sensitivity and specificity in detecting M. bovis strains (100.0%). Conclusions: PCR pncA-RFLP and RD-PCR represent very accurate and rapid tools to identify M. bovis. They can be easily implemented in each laboratory due to their low cost and easy use. Keywords: GenoType MTBC; lymph nodes; Mycobacterium bovis; PCR pncA-RFLP; RD-PCR.

PENGGUNAAN METODE PCR – RFLP (POLYMERASE CHAIN REACTION – RESTRICTION FRAGMENT LENGTH POLYMORFISM) DALAM MENDETEKSI JAMUR DERMATOFIT

ABSTRAK Dermatofitosis adalah salah satu jamur yang terdiri dari tiga genus : Epidermophyton, Trichophyton dan Microsporum. Penelitian ini bertujuan untuk mengidentifikasi jenis jamur dermatofit dengan metode PCR-RFLP. Penelitian ini merupakan penelitian observasional laboratorium dengan dengan menguji 23 sampel yang diperoleh dari beberapa klinik dan sekolah dasar di Makassar. Hasil penelitian menunjukkan bahwa semua sampel teridentifikasi yakni M. canis 26,1%, Trichophyton rubrum 13,1%,T. mentagrophytes 21,6%, T. tonsurans 8,7%, T. verrucosum 4,2% dan spesies unclassified 26,1%. Kami menyarankan Teknik PCR-RFLP dapat digunakan untuk konfirmasi jenis jamur dermatofit. Kata Kunci : Dermatofitosis, Jamur Dermatofit, PCR – RFLP

Identification of morphologically challenging Delia (Diptera: Anthomyiidae) species from field vegetable crops using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP)

Root feeding by the larvae of multiple Delia species can lead to economic loss in many agricultural crops. Field vegetables are subject to infestations by a species complex composed of Delia radicum (Linnaeus) (Diptera: Anthomyiidae), a pest in brassica crops (Brassicaceae), Delia antiqua (Meigen), believed to cause the majority of crop damage in onions, and the generalists Delia florilega (Zetterstedt), Delia platura (Meigen), and Botanophila fugax (Meigen) (Diptera: Anthomyiidae). Correct species identification is necessary to implement field management strategies, but these species are challenging to identify morphologically. We propose a polymerase chain reaction–restriction fragment length polymorphism method as a molecular tool to distinguish between five species of Delia and between two genetic lines of D. platura. The mitochondrial DNA cytochrome c oxidase subunit 1 barcode fragment is targeted, then the polymerase chain reaction product digested with four different restriction enzymes (AccI, BsrI, MlyI, and StyI). The BsrI enzyme distinguishes the two genetic lines of D. platura and D. florilega. The MlyI enzyme identifies B. fugax from the Delia species. Combining BsrI, StyI, AccI, and MlyI into double digestion reactions allows for rapid diagnostics among the species tested. Our method was validated using DNA from specimens collected in eastern Canada. This method provides tools in ecological and environmental studies where these species are of interest.

Complete Coding Genome Sequence of a Novel Porcine Reproductive and Respiratory Syndrome Virus 2 Restriction Fragment Length Polymorphism 1-4-4 Lineage 1C Variant Identified in Iowa, USA

A porcine reproductive and respiratory syndrome virus 2 strain was identified in lung samples from nursery piglets associated with a 17.15% mortality rate on a swine farm in Iowa, USA. Open reading frame 5 (ORF5) sequencing indicated that this strain is a restriction fragment length polymorphism (RFLP) 1-4-4 lineage 1C variant strain, and its complete coding genome sequence was determined.

Improvement in Group Identification of Dojo Loach, Misgurnus Anguillicaudatus, Using PCR-Restriction Fragment Length Polymorphism

Most wild types of dojo loach (Misgurnus anguillicaudatus) are gonochoristic diploids that are genetically diversified groups (A and B, further subdivided into B1 and B2), while clonal lineages inhabit certain localities in Japan. Through a series of genetic studies including DNA markers, the clonal loaches were deemed to originate from a hybridization event(s) between the A and B1 groups. However, combined analyses with other DNA markers are needed to identify each genetic group. In this study, we improved the PCR-restriction fragment length polymorphism (RFLP) analysis of the recombination activating gene 1 (RAG1) gene using digestion with two restriction enzymes, PvuII and StuI. The improved RAG1-RFLP analysis showed different fragment patterns for each group: two fragments (245 and 198 bp) for group A, three fragments (198, 147, and 98 bp) for group B1, and a single fragment (443 bp) for group B2. The clonal loaches exhibited four fragments (245, 198, 147, and 98 bp) derived from both groups A and B1. Moreover, the DNA markers were able to detect two different hybrid genotypes (A × B2 and B1 × B2). Thus, the improved RAG1-RFLP markers allowed for quick and accurate group identification of the dojo loaches.

WALTER: an easy way to online evaluate telomere lengths from terminal restriction fragment analysis

Background Telomeres, nucleoprotein structures comprising short tandem repeats and delimiting the ends of linear eukaryotic chromosomes, play an important role in the maintenance of genome stability. Therefore, the determination of the length of telomeres is of high importance for many studies. Over the last years, new methods for the analysis of the length of telomeres have been developed, including those based on PCR or analysis of NGS data. Despite that, terminal restriction fragment (TRF) method remains the gold standard to this day. However, this method lacks universally accepted and precise tool capable to analyse and statistically evaluate TRF results. Results To standardize the processing of TRF results, we have developed WALTER, an online toolset allowing rapid, reproducible, and user-friendly analysis including statistical evaluation of the data. Given its web-based nature, it provides an easily accessible way to analyse TRF data without any need to install additional software. Conclusions WALTER represents a major upgrade from currently available tools for the image processing of TRF scans. This toolset enables a rapid, highly reproducible, and user-friendly evaluation of almost any TRF scan including in-house statistical evaluation of the data. WALTER platform together with user manual describing the evaluation of TRF scans in detail and presenting tips and troubleshooting, as well as test data to demo the software are available at https://www.ceitec.eu/chromatin-molecular-complexes-jiri-fajkus/rg51/tab?tabId=125#WALTER and the source code at https://github.com/mlyc93/WALTER.