Rapid porcine detection in gelatin-based highly processed products using loop mediated isothermal amplification.

Low DNA concentration recovered from highly processed products such as gelatin and gelatin-based products renders difficulty in detecting porcine contamination using conventional PCR techniques. We documented here a porcine-specific loop-mediated isothermal amplification (LAMP) to identify porcine traces in gelatin products. The porcine-specific primers were designed according to mitochondrial DNA of Cytochrome b gene sequence. Here we used two different reaction mixtures for LAMP assay (GENIE and MYRM) against the same DNA samples extracted from gelatin products and porcine-specific primers to detect the presence of porcine DNA. The porcine-specific primers were shown to be specific only to Sus scrofa against 14 DNA of other meat species. The analytical sensitivity of the LAMP assay for porcine DNA detection is 1 pg/µL using both GENIE (within 30 m) and MYRM (within 60 m) reaction mixtures. Analysis against 32 samples of gelatin products showed that five samples were found to contain porcine DNA; two samples out of six gelatin powder samples and three gelatin capsule samples out of nine. Out of these five positive samples, three were not labeled containing porcine gelatin. Overall, LAMP assay in this study showed an excellent specificity, sensitivity and rapidity in detection of porcine DNA in gelatin products. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s13197-020-04932-2).

Role of miRNA in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Evidence suggests that miRNAs play an important role in progression, recurrence, metastasis and postoperative survival of HNSCC. Studies have investigated the utility of miRNAs as diagnostic/prognostic tools and as potential therapeutic targets and biomarkers that may improve the management and outcomes of HNSCC. The aim of this article is to review the current literature on aberrant expression profiles of miRNAs in biopsy samples of HNSCC and their role in cancer development, metastasis, prognosis and survival of these patients. This review gives an overview that miRNAs deregulation play major role in the development of HNSCC. They offer the potential to be used as biomarkers or novel therapeutic targets. Future research is required to test their use in both of these fields.

Haplotypes and variable position detection in the mitochondrial DNA coding region encompassing nucleotide positions 10,716-11,184.

This study evaluates the mitochondrial noncoding regions by using the Sanger sequencing method for application in Forensic Science. FTA® Technology (FTA™ paper DNA extraction) was utilized to extract DNA. Portion of coding region encompassing positions from (10,716 to 11,184) amplified in accordance with the Anderson reference sequence. PCR products purified by EZ-10 spin column were then sequenced and detected using the ABI 3730 × L DNA Analyzer. A new polymorphic positions 10,750 and 10,790 that are described may be suitable sources in future for identification purpose. The data obtained can be used to identify variable nucleotide positions characterized by frequent occurrence, most promising for identification variants.

pncA Mutations in the Specimens from Extrapulmonary Tuberculosis.

BACKGROUND: Pyrazinamide (PZA) is an effective antitubercular drug that becomes toxic to Mycobacterium tuberculosis when converted to pyrazinoic acid by pyrazinamidase (PZase), encoded by mycobacterial pncA. A strong association was noted between the loss of PZase activity and PZA resistance. The causative organisms in extrapulmonary tuberculosis are rarely cultured and isolated. To detect pncA mutations in specimens from extrapulmonary tuberculosis as confirmative diagnosis of mycobacterial infection and alternative susceptibility test to PZA. METHODS: Specimens were collected from clinically proven extrapulmonary tuberculosis. pncA was sequenced and compared with wild-type pncA. RESULTS: pncA from 30 specimens from 23 donors were successfully amplified (56.6% in specimens, 59% in donors). Six mutations in pncA were detected (20.0% in amplified specimens, 26.1% in specimen donors) at nucleotide positions of 169, 248 and 419. The mutation at position 169 results in substitution of aspartic acid for histidine, a possible allelic variation of M. bovis that have intrinsic PZA resistance. The mutation at position 248 changes proline into arginine and that at position 419, arginine into histidine. CONCLUSION: DNA-based diagnosis using pncA may be simultaneously useful for the early diagnosis of mycobacterial infection and the rapid susceptibility to PZA in extrapulmonary tuberculosis. A potential implication of pncA allelic variation at 169 might be suggested as a rapid diagnostic test for M. bovis infection or Bacille Calmette-Guérin (BCG) reactivation.

Characterization and antimicrobial activities of two Streptomyces isolates from soil in the periphery of Universiti Putra Malaysia.

This study was to assess the identification and antimicrobial activities of two actinomycete isolates. The two isolates designated as B8 and C2, were isolated from a patch of soil in the peripheral area of Universiti Putra Malaysia by streaking on starch casein agar after standard serial dilution procedures. Their antimicrobial activities were first evaluated against eight clinical laboratory strains namely Bacillus sp., Enterococcus sp., Escherichia coli, Klebsiella sp., Pseudomonas sp., Salmonella sp., Staphylococcus aureus, and Staphylococcus epidermidis by perpendicular streak method on Mueller Hinton and Tryptic Soy agar. In both media, a broad-spectrum antibacterial activity was observed for both isolates, with B8 against all the test bacteria and C2 against five of them (Bacillus sp., E. coli, Pseudomonas sp., S. aureus and S. epidermidis). Re-assessment against E. coli ATCC 25922 and S. aureus ATCC 25923 strains by similar method showed antibacterial activities by isolate B8 against both ATTC strains while C2 only against S. aureus ATCC 25923. Streptomyces griseus ATCC 10137 was included in the later experiment and showed antibacterial activity against both ATCC strains. Subsequently, the two isolates were identified by PCR/sequencing techniques and phylogenetic analysis to be Streptomyces species (>93% homology based on 16S rRNA and rpoB genes). Characterization on cultural characteristic and viable count at different temperatures (37ºC and 28ºC), on different microbiological media (AIA, ISP-2, MHA, NA, PDA and TSA), were performed. More morphological features were observed on ISP-2 for both isolates. A higher growth yield was also observed at 28ºC in all media but in comparing that between the two isolates, isolate B8 outnumbered C2 at all experimental conditions. The observed variation in cultural traits and growth yield indicate unique properties between the two antibiotic-producing isolates.

Evaluation of Helicobacter pylori vacA genotypes in Iranian patients with peptic ulcer disease.

Helicobacter pylori is the major cause of active chronic gastritis and peptic ulcers in humans and has been linked to gastric carcinoma and lymphoma. The vacuolating cytotoxin vacA and cag pathogenicity island (cag PAI) are two identified virulence factors that are considered to have an important role in the pathogenesis of H. pylori infection. The aim of this study is to investigate the H. pylori vacA alleles in Iranian patients with peptic ulcer disease. In order to investigate this, biopsy specimens were obtained from 84 patients with gastric ulcer, gastritis, and duodenal ulcer. DNA extraction and PCR were used to detect the presence or absence of glmM, cagA and to assess the polymorphism of vacA. Of the 77 glmM PCR-positive biopsy specimens, 55 (71%) had the vacA signal sequence genotype s1, and 22 (29%) had subtype s2. vacA mid-region analysis revealed that 31 (40%) were vacA m1 and 46 (60%) were m2. The presence of the cagA gene correlated with vacA signal sequence type s1, whereas type s2 was predominantly found in cagA-negative samples (P < 0.001). Thus, the detection of vacA and cagA, virulence markers described in several clinical outcomes may be used to help the treatment and prevention of H. pylori in Iran.

Detection of tdh and trh genes in Vibrio parahaemolyticus isolated from Corbicula moltkiana prime in West Sumatera, Indonesia.

The occurrence of Vibrio parahaemolyticus in raw Corbicula moltkiana Prime from Lake Singkarak and Pasar Raya Padang market and in cooked samples in West Sumatera, Indonesia, was studied. Thirteen raw and seven cooked bivalve samples were positive using CHROMAgar Vibrio medium. All 47 V parahaemolyticus isolates were positive for toxR gene but negative for trh. However, 36% (17/47) of V parahaemolyticus strains were positive for tdh gene. Antibiotic profiling showed that 76% and 38% of isolates from raw and cooked bivalves respectively were resistant to ampicillin. Using RAPD-PCR analysis, most of the strains were clustered according to their source of isolation but some of the strains from raw and cooked samples were clustered together. These results indicate that pathogenic V parahaemolyticus isolates are present in Corbicula moltkiana Prime in West Sumatera, Indonesia, suggesting that V parahaemolyticus may also be present in seafood in other regions of Indonesia.

Preliminary screening of endophytic fungi from medicinal plants in malaysia for antimicrobial and antitumor activity.

The screening of antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi was carried out on isopropanol extracts prepared from 121 isolates of endophytic fungi isolated from medicinal plants in Malaysia. Sensitivity was found to vary among the microorganisms. Bacillus subtilis, Saccharomyces cerevisiae and Alternaria sp. were susceptible to extracts from three, two and two isolates of endophytic fungi, respectively. None were found effective against Salmonella typhimurium. Sixteen endophytic fungal isolates tested were also found to exhibit antitumor activity in the yeast cell-based assay.