Practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a South African setting

Background: Formalin-fixed paraffin-embedded (FFPE) tissue archives in hospitals, biobanks, and others offer a vast collection of extensive, readily available specimens for molecular testing. Unfortunately, the use of tissue samples for molecular diagnostic applications is challenging; thus, the forensic pathology FFPE tissue archives in Africa have been a largely unexploited genetic resource, with the usability of DNA obtainable from these samples being unknown.Intervention: The study, conducted from January 2015 to August 2016, determined the usefulness of FFPE tissue as a reliable source of genetic material for successful post-mortem molecular applications and diagnostics. Formalin-fixed paraffin-embedded tissue samples were collected and archived from autopsies conducted over 13 years in the forensic medicine department of the University of Pretoria (Pretoria, South Africa). Deoxyribonucleic acid from FFPE tissue samples and control blood samples was amplified by high-resolution melt real-time polymerase chain reaction before sequencing. The procurement parameters and fixation times were compared with the quantity and quality of the extracted DNA and the efficiency of its subsequent molecular applications.Lessons learnt: This study has shown that FFPE samples are still usable in molecular forensics, despite inadequate sample preparation, and offer immense value to forensic molecular diagnostics.Recommendations: FFPE samples fixed in formalin for more than 24 h should still be used in molecular diagnostics or research, as long as the primer design targets amplicons not exceeding 300 base pairs.

Microbial additives in the composting process / Aditivos microbianos no processo de compostagem

ABSTRACT Composting is the process of natural degradation of organic matter carried out by environmental microorganisms whose metabolic activities cause the mineralization and partial humification of substances in the pile. This compost can be beneficially applied to the soil as organic fertilizer in horticulture and agriculture. The number of studies involving microbial inoculants has been growing, and they aim to improve processes such as composting. However, the behavior of these inoculants and other microorganisms during the composting process have not yet been described. In this context, this work aimed to investigate the effects of using a microbial inoculum that can improve the composting process and to follow the bacterial population dynamics throughout the process using the high-resolution melt (HRM) technique. To do so, we analysed four compost piles inoculated with Bacillus cereus, Bacillus megaterium, B. cereus + B. megaterium and a control with no inoculum. The analyses were carried out using samples collected at different stages of the process (5th to 110th days). The results showed that the bacterial inocula influenced the process of composting, altering the breakdown of cellulose and hemicelluloses and causing alterations to the temperature and nitrogen levels throughout the composting process. The use of a universal primer (rDNA 16S) allowed to follow the microbial succession during the process. However, the design of a specific primer is necessary to follow the inoculum throughout the composting process with more accuracy.

RESUMO A compostagem é um processo de degradação natural da matéria orgânica realizado por microrganismos presentes no ambiente, levando a mineralização e humificação parcial das substâncias presentes na pilha, esse composto formado pode ser beneficamente aplicado ao solo como fertilizante orgânico na horticultura e agricultura. O número de estudos envolvendo inoculantes microbianos é crescente, os quais tem por objetivo a otimização de processos de compostagem. Contudo, o comportamento desses inoculantes e da microbiota ao longo do processo não tem sido caracterizado. Nesse contexto, este trabalho foi realizado com o objetivo de avaliar o efeito da utilização de um inóculo bacteriano que promova melhorias no processo de compostagem, bem como o de acompanhar a dinâmica populacional bacteriana ao longo de todo o processo através da técnica de High Resolution Melt (HRM). Para isso foram analisados quatro pilhas de compostagem inoculadas com Bacillus cereus, Bacillus megaterium, B. cereus + B. megaterium e o controle sem adição de inóculo. Foram realizadas análises químicas e moleculares (HRM) das amostras coletadas em diferentes períodos da compostagem (5º ao 110º dias). Os resultados mostraram que os inóculos bacterianos influenciaram no processo de compostagem com alteração na degradação de celulose, hemicelulose bem como alteração da temperatura e níveis de nitrogênio ao longo da compostagem. A utilização de um primer universal (rDNA 16S) permitiu acompanhar a sucessão bacteriana ao longo do processo, nos tratamentos. Contudo a construção de um primer específico é necessário para acompanhar de maneira mais precisa o inóculo durante o desenvolvimento da compostagem.

Comparison of PCR and HRM for detecting bacterial drug resistance gene aac (6′)- Ib-cr / 国际检验医学杂志

Objective To study the difference of detection rate between PCR and high-resolution melt (HRM)for detecting the bacterial drug resistance gene aac(6′)-Ib-cr.Methods The PCR method was adopted to detect the aac(6′)-Ib gene in 299 strains of common Gram-negative bacilli,and the PCR positive products of aac (6′)-Ib were digested with BtsCI.Meanwhile Aac (6′)-Ib and aac (6′)-Ib-cr genes in above bacterial strains were detected by adopting the HRM technique.Results A total of 29 isolates were i-dentified as aac (6′)-Ib gene,and 21 mutations were aac (6′)-Ib-cr,with a mutation rate of 72.4% (21/29)for PCR and restriction enzyme digestion.And the results of the HRM test were in good agreement with each other.Conclusion PCR and HRM are similar for the detection of the bacterial resistance gene aac (6′)-Ib-cr,and aac (6′)-Ib-cr gene could be detected by a simpler HRM tech-nique instead of PCR and enzyme digestion,especially for a large number of screening.

Applying high resolution melt analysis to discriminate VEB-3 hypotype of the clinical gram negative isolates / 中华微生物学和免疫学杂志

Objective To establish a new method, applying high resolution melt, to discriminate the VEB-3 hypotype from the clinical gram negative isolates. Methods From January to December 2003,292 consecutive and non-repetitive gram-negative bacteria producing VEB extended spectrum β-lactamase (ESBL) were collected. Extract the DNA of clinical gram negative isolates with phenol-chloroform. PCR was performed to amplify the VEB gene with the DNA being template. After that, we amplify the fragment of VEB gene containing the position 168. Then we detect the high resolution melt curve and analyze them. At last, we analyze the results of sequence and high resolution melt( HRM ). Results VEB-1 and VEB-3 gene are markedly different through HRM analysis. Conclusion It is accurately and quickly for us to identify the VEB-3 from other hypotype through the technology of HRM.