A low-cost PCR instrument for molecular disease diagnostics based on customized printed circuit board heaters.

This article describes the fabrication of a low-cost Polymerase Chain Reaction (PCR) instrument to detect diseases. In order to reduce the instrument price and simplify construction we developed an alternative fabrication process, transforming conventional printed circuit boards (PCB) in heating elements, avoiding the use of aluminum heating/cooling blocks and Peltier devices. To cool down the reaction a simple computer fan was used. The vial holder was fabricated using two double side PCB boards assembled in a sandwich-like configuration. The bottom PCB has a resistance of 0.9 Ω used to heat the reaction mix, while the top layer has a resistance of 1.1 Ω to heat the vial body, preventing vapor condensation. The top board was maintained at ~ 110 ± 1 °C during all cycles. The final device was able to heat and cool down the reaction at rates of ~ 2.0 °C/s, a rate comparable to commercial thermocyclers. An SMD NTC thermistor was used as temperature sensors, and a PID (proportional-integral-derivative) control algorithm was implemented to acquire and precisely control the temperature. We also discuss how the instrument is calibrated. The device was tested successfully for the amplification of T. pallidum (Syphilis) bacterial DNA and Zika virus RNA samples, showing similar performance to a commercial PCR instrument.

Use of Escherichia coli BOX-PCR fingerprints to identify sources of fecal contamination of water bodies in the State of São Paulo, Brazil.

Repetitive element sequence-based polymerase chain reaction (rep-PCR) is one of the commonest methods used to identify sources of fecal contamination of water systems. In this work, BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) was used to discriminate Escherichia coli strains originating from different animals and water sources, and the suitability of the technique for bacterial source tracking (BST) was evaluated. A total of 214 strains from humans, 150 strains from animals, 55 strains from sewage and 77 strains from water bodies were analyzed by the BOX-PCR technique. When maximum similarity between the fingerprints was used, a correct classification rate of 84% was achieved for strains from human and animal sources. Furthermore, 95% of the strains found in sewage were classified as being from human sources by at least one of the four classification tools used. Classification of the strains found in water bodies in the State of São Paulo was based on the fingerprints obtained for human and animal sources. Most of the sampling sites appeared to be affected by mixed sources of fecal contamination. The use of BOX-PCR for BST could be especially valuable in developing countries, where simplicity and cost are important considerations.

Development, verification, and validation of an RT-qPCR-based protocol for Yellow Fever diagnosis.

INTRODUCTION: Yellow fever (YF) is a public health threat with frequent outbreaks in tropical and subtropical areas, despite the existence of a safe and effective vaccine. The diagnosis of acute infection of the etiologic agent relies mainly on real-time reverse transcription-polymerase chain reaction (RT-qPCR)-based assays. OBJECTIVES: The aim of this study was to evaluate and compare this novel protocol for yellow fever virus (YFV) diagnosis against assays developed in-house by reference laboratories for arboviruses. METHODS: We developed a novel molecular protocol for the detection of YFV that includes an Internal Control to validate the reaction and an External Control to monitor the RNA extraction efficiency. RESULTS AND DISCUSSION: Our assay detects one viral genome per reaction and displays no cross-reactions with dengue (1-4), Zika, or Chikungunya viruses. This novel assay yielded 95% of agreement with the reference method recommended by the Pan American Health Organization when analyzing 204 clinical samples and cultured viruses, these samples were analyzed in 3 different diagnosis centers for arboviruses in Brazil. The data suggest the use of the proposed multiplex assay protocol to do routine tests in a clinical laboratory. This product adds higher specificity and sensitivity in addition to reduced cost per test due to hands-on time and reagent spending.

Ferric iron uptake genes are differentially expressed in the presence of copper sulfides in Acidithiobacillus ferrooxidans strain LR.

Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide ores. This work aimed to investigate the relative expression of genes related to the iron uptake system when A. ferrooxidans LR was maintained in contact with chalcopyrite or bornite as the sole energy source. Real-time quantitative PCR analysis revealed that the presence of bornite had no effect on the expression of seven genes related to the siderophore-mediated Fe(III) uptake system, while in the presence of chalcopyrite the expression of the genes was up-regulated. Bioinformatic analysis of the genomic region where these genes were found revealed the existence of three new putative DNA-binding sequences for the ferric iron uptake transcriptional regulator (Fur). Electrophoretic mobility shift assays demonstrated that a purified A. ferrooxidans His-tagged Fur protein was able to bind in vitro to each of these putative Fur boxes, suggesting that Fur regulated the expression of these genes. The expression of fur and two known Fur-regulated genes, mntH and dsrK, was also investigated in the presence of chalcopyrite. While the expression of fur and mntH was up-regulated, the expression of dsrK was down-regulated. The low amount of ferrous iron in the medium was probably responsible for the up-regulation of fur and the genes related to the siderophore-mediated Fe(III) uptake system when A. ferrooxidans LR was kept in the presence of chalcopyrite. A homology model of the A. ferrooxidans Fur was constructed and revealed that the putative DNA-binding surface presents conserved positively charged residues, supporting a previously suggested mode of interaction with DNA. The up-regulation of fur and the siderophore-mediated Fe(III) uptake genes, and the down-regulation of dsrK suggest that in the presence of chalcopyrite Fur acts as a transcription inducer and repressor.

Prevalence of virulence factors in Escherichia coli isolated from healthy animals and water sources in Brazil.

The aim of this work was to verify the presence of seven virulence factors (ST, LT, eae, stx(1), stx(2), INV and EAEC) among Escherichia coli strains isolated from healthy humans, bovines, chickens, sheep, pigs and goats, from two sewage treatment plants and from the Tietê River. We have found a high prevalence of eae, stx(1) and stx(2) in ruminants. The EAEC gene was only found in humans and sewage. No strains presented ST, LT or INV. BOX-PCR fingerprints revealed a high diversity among the strains analysed and a non-clonal origin of strains that presented the same virulence factors. Therefore, we concluded that ruminants may constitute an important reservoir of most diarrheagenic E. coli in Brazil, except for EAEC strains. These results emphasize the importance of the identification of the animal source of fecal contamination for the correct water risk assessment.

Gene expression modulation by chalcopyrite and bornite in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a mesophilic, acidophilic, chemolithoautotrophic bacterium that obtains energy from the oxidation of ferrous iron (Fe2+), elemental sulfur and reduced sulfur compounds. The industrial interest in A. ferrooxidans resides in its capacity to oxidize insoluble metal sulfides into soluble metal sulfates, thus allowing the recovery of the desired metals from low-grade sulfide ores. In the present work, RNA arbitrarily primed PCR (RAP-PCR) was performed to identify cDNAs differentially expressed in A. ferrooxidans cells grown in the presence of Fe2+ and cells maintained for 24 h in the presence of the copper sulfides bornite and chalcopyrite. Eighteen cDNAs corresponding to genes with known function were identified, and their relative expression was further characterized by real-time quantitative PCR. Bornite had a mild effect on the expression of the 18 genes analyzed. None of these genes was down-regulated and among the few genes up-regulated, it is worth mentioning lepA and def-2 that are involved in protein synthesis. Chalcopyrite presented the most significant changes. Five genes related to protein processing were down-regulated, and another 5 genes related to the transport system were up-regulated. The up- and down-regulation of these genes in the presence of bornite and chalcopyrite could be due to alterations in the ideal pH, presence of copper ions in solution and nutrient limitation. The results suggest that gene expression modulation might be important for the A. ferrooxidans early response to copper sulfides.

Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment.

Hearing impairment is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non-syndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafness-associated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced.

Connexin mutations in Brazilian patients with skin disorders with or without hearing loss.

The connexins are a family of proteins whose major function is as part of the gap junctions of cell-to-cell channels. They are expressed in several tissues including brain, skin, and cochlea. Mutations in connexin genes play a major role in non-syndromic sensorineural deafness, but have been also described in individuals with variable dermatological features. In recent years, many genes responsible for hereditary skin diseases have been discovered. These genes may encode different proteins that participate in the terminal differentiation of the epidermis. Therefore alteration or absence of these proteins causes a keratinization disorder. It has been demonstrated that distinct germline mutations within six connexin (Cx) genes GJB2 (Cx26), GJB6 (Cx30), GJB3 (Cx31), GJA1 (Cx43), GJB4 (Cx30.3), and GJB5 (Cx31.1), may cause sensorineural hearing loss and various skin disease phenotypes. The crucial functional importance of each of these connexins in the mentioned ectodermic tissues is reflected by the finding that genetic defects in their genes produce a wide spectrum of genetic disorders comprising sensorineural hearing loss, disorders of cornification of the skin, hair, and nails, and keratitis. Here, we report on different mutations in the connexin genes in individuals with or without hearing loss and different skin disorders illustrating the clinical and genetic heterogeneity of the condition.

Type II autosomal recessive cutis laxa: report of another patient and molecular studies concerning three candidate genes.

Cutis laxa is a rare disorder of connective tissue in which the skin sags excessively, giving the individual an aged appearance. In the present study we analyzed three unrelated families with type II autosomal recessive cutis laxa for mutations in three genes implicated in other forms of cutis laxa; LOX, FBLN4, and FBLN5 genes. Two individuals have been previously reported, and the third case is described in detail. No causative mutations were identified.

Conventional culture method and qPCR using 16S rDNA for tissue bank: a comparison using a model of cardiac tissue contamination.

Real-time polymerase chain reaction (qPCR) using 16S rDNA is an alternative to conventional culture-based tests. The aim of this study was to compare the conventional culture method with qPCR using 16S rDNA in a model of cardiac tissue contamination. Samples of cardiac tissue for artificial contamination with Escherichia coli and control samples were submitted for DNA extraction, which was conducted by selective and alkaline lysis and purification steps. A standard curve for 16S rDNA was constructed to determine the efficiency and analytical sensitivity of the assay in concentrations from 106 to 102 c.f.u. ml-1 using TaqMan Master Mix. 16S rDNA was detected in all contaminated samples; however, it was not detected in the the final washing step solution of the sample with a bioburden of 102 c.f.u. ml-1. Using qPCR is a potential alternative to conventional culture for microbiological safety testing of allograft tissues for biobanking, reducing the time and labour input required.

Molecular findings in Brazilian patients with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder of increased bone fragility and low bone mass. Severity varies widely, ranging from intrauterine fractures and perinatal lethality to very mild forms without fractures. Most patients with a clinical diagnosis of OI have a mutation in the COL1A1 or COL1A2 genes that encode the a chains of type I procollagen, the major protein in bones. Hence, the aim of the present study was to identify mutations in the COL1A1 gene in 13 unrelated Brazilian OI patients. This is the first molecular study of OI in Brazil. We found 6 mutations, 4 of them novel (c.1885delG, p.P239A, p.G592S, p.G649D) and 2 previously described (p.R237X and p.G382S). Thus, the findings show that there are no prevalent mutations in our sample, and that their distribution is similar to that reported by other authors, with preponderance of substitutions for glycine in the triple helix domain, causing OI types II, III and IV.

[Prospects for genetic hearing loss screening: 35delG mutation tracking in a newborn population]. / Perspectivas para triagem da deficiência auditiva genética: rastreamento da mutação 35delG em neonatos.

OBJECTIVES: To investigate the prevalence of the 35delG mutation in a newborn population, with specific molecular testing, and to evaluate the prospects for genetic neonatal screening for hearing impairment. POPULATION AND METHOD: 233 newborn were evaluated at the Hospital de Base de São José do Rio Preto, SP, for molecular analysis of the 35delG mutation in the connexin 26 gene, with the reaction technique in allele-specific polymerase chain reaction, after genomic DNA extraction from umbilical cord blood. RESULTS: Five heterozygotes were identified, obtaining a prevalence of 2.24% of 35delG mutation carriers in the study population. CONCLUSION: Using the molecular test allowed for the identification of the 35delG mutation in the study population with the possibility of being used as a complement to neonatal audiometric screening as being simple, fast, and easily to perform with low costs.

Molecular genetics of non-syndromic deafness.

One in every 1,000 newborn suffers from congenital hearing impairment. More than 60% of the congenital cases are caused by genetic factors. In most cases, hearing loss is a multifactorial disorder caused by both genetic and environmental factors. Molecular genetics of deafness has experienced remarkable progress in the last decade. Genes responsible for hereditary hearing impairment are being mapped and cloned progressively. This review focuses on non-syndromic hearing loss, since the gene involved in this type of hearing loss have only recently begun to be identified.

Screening for mutations in the GJB3 gene in Brazilian patients with nonsyndromic deafness.

Deafness is a complex disorder that is affected by a high number of genes and environmental factors. Recently, enormous progress has been made in nonsyndromic deafness research, with the identification of 90 loci and 33 nuclear and 2 mitochondrial genes involved (http://dnalab-www.uia.ac.be/dnalab/hhh/). Mutations in the GJB3 gene, encoding the gap junction protein connexin 31 (Cx31), have been pathogenically linked to erythrokeratodermia variabilis and nonsyndromic autosomal recessive or dominant hereditary hearing impairment. To determine the contribution of the GJB3 gene to sporadic deafness, we analysed the GJB3 gene in 67 families with nonsyndromic hearing impairment. A single coding exon of the GJB3 gene was amplified from genomic DNA and then sequenced. Here we report on three amino acid changes: Y177D (c.529T > G), 49delK (c.1227C > T), and R32W (c.144-146delGAA). The latter substitution has been previously described, but its involvement in hearing impairment remains uncertain. We hypothesize that mutations in the GJB3 gene are an infrequent cause of nonsyndromic deafness.

Newborn hearing screening and genetic testing in 8974 Brazilian neonates.

OBJECTIVE: An early diagnosis has been a priority in the audiological practice. Identifying hearing loss until 3 months old through Universal Newborn Hearing Screening and intervention before 6 months old, minimize the impact of auditory loss in the health and communication development of these children. However, in the clinical practice, despite the help of the risk indicators in the audiological and etiological diagnosis, the integrated services have come up against the challenge of determining the causes of auditory loss, bearing in mind that approximately 50% of the subjects who have congenital loss do not show risk factors in their clinical history. The current research aims introduce together etiologic and audiological diagnosis of newborns. METHODS: We eluted dried blood spots from paper and performed genetic testing for 35delG mutation in 8974 newborns that were also screened for transient otoacoustic emissions (TOAE). In addition, the A1555G and A827G mutations in the MTRNR1 mitochondrial gene were screened in all newborns. RESULTS: We have found 17 individuals who failed in TOAE. Among them, we detected 4 homozygous newborns for 35delG mutation and 3 individuals with A827G mutation in the MTRNR1 mitochondrial gene. The frequency of 35delG carriers was 0.94% [84/8974]. In all 17 individuals who failed in OAE no other mutation besides those mentioned above was found. CONCLUSIONS: The results greatly contribute to the public health area indicating the etiologic diagnosis, allowing family counseling as well as the early rehabilitation treatment or surgical intervention. Over time that will help to reduce the costs of rehabilitation considerably.

Molecular genetics study of deafness in Brazil: 8-year experience.

Hereditary hearing loss is a complex disorder that involves a large number of genes. In developed countries, 1 in 1,000 children is born with deafness severe enough to require special education services, and about 60% of the cases of isolated deafness have a genetic origin. Although more than 100 genes for hearing loss are known currently, only a few are routinely tested in the clinical practice. In this study, we present our findings from the molecular diagnostic screening of the GJB2 and GJB3 genes, del(GJB6-D13S1,830) and del(GJB6-D13S1,854) deletions in the GJB6 gene, Q829X mutation in the otoferlin gene (OTOF) and, the A1,555G and A7,445G mutations in the mitochondrial genome over an 8-year period. Mutations analysis in the previously mentioned genes and mutations was performed on 645 unrelated Brazilian patients with hearing loss who fell into two different testing groups. Different mutations in the GJB2 gene were responsible for most of cases studied, but deletions in the GJB6 gene as well as mitochondrial mutations were also found. While most cases of hearing loss in this country are due to environmental factors, the genetic etiology of deafness will increasingly be determined as more genetic tests become available.

Molecular study in Brazilian cochlear implant recipients.

The most common form of non-syndromic autosomal recessive deafness (NSRD) is caused by mutations in the GJB2 gene. Recently, a deletion truncating the GJB6 gene, called del(GJB6-D13S1,830) has also been described normally accompanying mutations in another allele of the GJB2 gene. Among all the mutations described to date, 35delG in the GJB2 gene is the most common. Preliminary data suggest that pathologic changes due to GJB2 mutations do not affect the spiral ganglion cells, which are the site of stimulation of the cochlear implant. Besides, the survival of the spiral ganglion cells is believed to be an important determinant of the outcome after surgery. Therefore, we have studied 49 non-syndromic deaf patients with unknown etiologies in order to determine the prevalence of GJB2 and GJB6 gene mutations in patients undergoing cochlear implantation surgery. Also, the molecular studies were performed using polymerase chain reaction amplification and direct sequencing. As a result, we found 19 individuals with GJB2 mutation including one new mutation (K168R), one patient homozygous for the del(GJB6-D13S1,830). These results establish that genetic screening can provide an etiologic diagnosis, and may help with prognosis after cochlear implantation, as has been hypothesized in previous studies.

Perspectivas para triagem da deficiência auditiva genética: rastreamento da mutação 35delG em neonatos / Prospects for genetic hearing loss screening: 35delG mutation tracking in a newborn population

OBJETIVO: Investigar a prevalência da mutação 35delG em amostra de recém-nascidos, com teste molecular específico; avaliar as perspectivas para a triagem neonatal genética para a deficiência auditiva. CASUíSTICA E MÉTODO: Foram avaliados 223 recém-nascidos no Hospital de Base de São José do Rio Preto, em São Paulo, para análise molecular da mutação 35delG, no gene da conexina 26, com a técnica da reação em cadeia da polimerase alelo-específico, após extração do DNA genômico de sangue de cordão umbilical. RESULTADOS: Foram identificados cinco heterozigotos, obtendo-se prevalência de 2,24 por cento de portadores da mutação 35delG, na população do estudo. CONCLUSAO: O uso do teste molecular permitiu a identificação da mutação 35delG na população do estudo, podendo ser utilizado como complemento aos rastreamentos audiométricos neonatais por ser simples, rápido, de fácil execução e de baixo custo.

Genética molecular da deficiência auditiva não-sindrômica / Molecular genetics of non-syndromic deafness

Aproximadamente 1/1000 recém-nascidos apresentam deficiência auditiva congênita, sendo 60 por cento dessas de etiologia genética. Na maioria dos casos, a deficiência auditiva é uma doenca multifatorial causada por ambos os fatores, genéticos e ambientais. A genética molecular da deficiência auditiva tem apresentado grandes avancos na última década, pois os genes responsáveis pela deficiência auditiva hereditária vêm sendo progressivamente mapeados e clonados. Esta revisão enfatiza a deficiência auditiva não-sindrômica, uma vez que, os genes envolvidos nesse tipo de deficiência foram identificados recentemente.