Isolation of total DNA from bacteria and yeast

Many procedures in molecular biology require the isolation of high quality genomic DNA. This study investigated a new method to extract DNA from Gram-negative, Gram-positive bacteria, Mycobacteria and yeasts. Guanidine thioisocyanate present in DNAzol is capable of binding DNA to silica particle column. Subsequently the silica with adsorbed DNA is washed to remove impurities and the clean DNA eluted in appropriate buffer. Results indicated that the new extraction method is simple and reproducible. This isolation technique is faster and easier to perform than the other conventional extraction methods. Finally the recovered DNA is of high quality and suitable for downstream applications. (African Journal of Biotechnology: 2003 2(8): 251-253

Isolation of total DNA from bacteria and yeast

Many procedures in molecular biology require the isolation of high quality genomic DNA. This study investigated a new method to extract DNA from Gram-negative, Gram-positive bacteria, Mycobacteria and yeasts. Guanidine thioisocyanate present in DNAzol is capable of binding DNA to silica particle column. Subsequently the silica with adsorbed DNA is washed to remove impurities and the clean DNA eluted in appropriate buffer. Results indicated that the new extraction method is simple and reproducible. This isolation technique is faster and easier to perform than the other conventional extraction methods. Finally the recovered DNA is of high quality and suitable for downstream applications. (African Journal of Biotechnology: 2003 2(8): 251-253

Real-time PCR assay for detection of Staphylococcus aureus, Panton-Valentine Leucocidin and Methicillin Resistance directly from clinical samples

Rapid detection of Methicillin Resistant Staphylococcus aureus (MRSA) is an important concern for both treatment and implementation of infection control policies. The present study provides an \u2018in house\u2019 real-time PCR assay to detect directly nuc, pvl, and mecA genes. The assay is able to perform identification of MRSA, Methicillin-Sensitive S. aureus, Methicillin-Resistant Coagulase Negative Staphylococci and the Panton-Valentine leukocidin virulence gene from rectal and pharyngeal swab samples in a screening context. We found an analytical sensitivity of this current Triplex PCR assay of 514 CFU/mL. Analytical specificity was tested with different Gram-positive and Gram-negative species and yielded no false-positive PCR signal. The sensitivity and specificity of the Triplex Real Time PCR were both 100% for these targets when compared with the culture and conventional methods. This assay is readily adaptable for routine use in a microbiology laboratory, as it will enable the implementation of timely and properly guided therapy and infection control strategies

Intrinsic role of coagulase negative staphylococci norA-like efflux system in fluoroquinolones resistance

NorA is a Staphylococcus aureus multidrug transporter that exports structurally distinct compounds including fluoroquinolones. In this study norA-like genes of Staphylococcus epidermidis (norASEP) and Staphylococcus haemolyticus (norASHAE) were identified and sequenced. The nucleotide identity of norASEP and norASHAE with norA was 75.3 and 74.1%, respectively, and the amino acid identity 87.7 and 86%, respectively. Inactivation of norASEP increased the ciprofloxacin susceptibility of E. coli DH5\u3b1 carrying the pB SK 198 norASEP EZ cat norASEP plasmid

Prevalence of and short-term changes in conjunctival manifestations among patients with SARS-CoV-2 infection

[no abstract available

CFTR Modulation Reduces SARS-CoV-2 Infection in Human Bronchial Epithelial Cells

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19

Development of a real-time quantitative polymerase chain reaction assay for the detection of congenital human cytomegalovirus infection in urine samples.

A TaqMan real-time quantitative PCR (qPCR), based on amplification of HCMV UL54 gene specific sequence, was developed and compared with shell vial viral culture assay, the gold standard technique for the diagnosis of congenital HCMV infection, using urine samples collected from 110 newborns. The results indicate that this qPCR is slightly more sensitive than shell vial assay suggesting that qPCR may be considered a useful alternative for diagnosing congenital HCMV infection

Short Communication - Isolation of total DNA from bacteria and yeast

Many procedures in molecular biology require the isolation of high quality genomic DNA. This study investigated a new method to extract DNA from Gram-negative, Gram-positive bacteria, Mycobacteria and yeasts. Guanidine thioisocyanate present in DNAzol is capable of binding DNA to silica particle column. Subsequently the silica with adsorbed DNA is washed to remove impurities and the clean DNA eluted in appropriate buffer. Results indicated that the new extraction method is simple and reproducible. This isolation technique is faster and easier to perform than the other conventional extraction methods. Finally the recovered DNA is of high quality and suitable for downstream applications

Modification of penicillin-binding protein 5 associated with high-level ampicillin resistance in Enterococcus faecium

High-level ampicillin resistance in Enterococcus faecium has been shown to be associated with the synthesis of a modified penicillin-binding protein 5 (PBP 5) which had apparently lost its penicillin-binding capability (R. Fontana, M. Aldegheri, M. Ligozzi, H. Lopez, A. Sucari, and G. Satta. Antimicrob. Agents Chemother. 38:1980-1983, 1994). The pbp5 gene of the highly resistant strain E. faecium 9439 was cloned and sequenced. The deduced amino acid sequence showed 77 and 54% homologies with the PBPs 5 of Enterococcus hirae and Enterococcus faecalis, respectively. A gene fragment coding for the C-terminal part of PBP 5 containing the penicillin-binding domain was also cloned from several E. faecium strains with different levels of ampicillin resistance. Sequence comparison revealed a few point mutations, some of which resulted in amino acid substitutions between SDN and KTG motifs in PBPs 5 of highly resistant strains. One of these converted a polar residue (the T residue at position 562 or 574) of PBP 5 produced by susceptible and moderately resistant strains into a nonpolar one (A or I). This alteration could be responsible for the altered phenotype of PBP 5 in highly resistant strains