A qPCR technology for direct quantification of methylation in untreated DNA.

DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.

Aplicación de PCR a partir de cultivo en la identificación de subespecies del complejo Mycobacterium Tuberculosis / The use of culture-based PCR for the identification of subspecies of the mycobacterium tuberculosis complex

Los métodos diagnósticos clásicos de tuberculosis (TB) se basan en la utilización de baciloscopía y cultivo. La identificación del agente etiológico desde la positivización del cultivo requiere entre 10 y 15 días, mientras que el empleo de la reacción en cadena de la polimerasa (PCR) disminuye el tiempo a 24 h, lo que permite no solo identificar las subespecies del complejo Mycobacterium tuberculosis (CMTB) sino también diferenciarlas de otras especies ambientales clínicamente importantes (MOTT) facilitando el diagnóstico y tratamiento. El objetivo del presente trabajo fue determinar la utilidad de la PCR en la identificación temprana de las micobacterias pertenecientes al CMTB, a partir de cultivos positivos, de pacientes con sospecha de TB, atendidos en un hospital pediátrico de alta complejidad, durante un período de cuatro años. A cada muestra, se le realizó baciloscopía y cultivo en medio líquido. A los cultivos positivos, una inmunocromatografía lateral (TBIDR) y luego PCR. El 4,6% del total de muestras (510/11.162) pertenecientes a 198 pacientes presentó cultivos positivos. Cuatrocientos veintiseis (84%) correspondieron a muestras respiratorias. El rendimiento de la baciloscopía directa fue del 41% (194/470). Cuatrocientos treinta y ocho (86%) resultaron M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG y 44 (9%) MOTT. La utilización de medios de cultivos líquidos junto con el empleo de PCR favorecen una rápida orientación microbiológica y constituye una estrategia útil para optimizar el manejo clínico de estas infecciones, desde el punto de vista terapéutico y epidemiológico, especialmente en pediatría (AU)

Classical diagnostic methods for tuberculosis (TB) are based on the use of smear microscopy and culture. The identification of the etiological agent from positive culture requires 10 to 15 days, while the use of the polymerase chain reaction (PCR) reduces the time to 24 h, which allows not only to identify the subspecies of the Mycobacterium tuberculosis complex (MTC) but also to differentiate them from clinically important environmental mycobacteria other than tuberculosis (MOTT), facilitating diagnosis and treatment. The aim of this study was to determine the usefulness of PCR in the early identification of mycobacteria belonging to the MTC, from positive cultures of patients with suspected TB seen in a pediatric tertiary hospital over a 4-year period. For each sample, smear microscopy and culture in liquid medium was performed. Positive cultures were subjected to lateral immunochromatography (TBIDR) and then PCR. Of the total number of samples (510/11,162) belonging to 198 patients, 4.6% showed positive cultures; 426 (84%) were respiratory samples. The direct smear microscopy yield was 41% (194/470). Overall, 438 (86%) were found to be M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG, and 44 (9%) MOTT. The use of liquid culture media together with the use of PCR favors a rapid microbiological orientation and is a useful strategy to optimize the clinical management of these infections, from a therapeutic and epidemiological point of view, especially in children (AU)

Lyophilization of Molecular Biology Reactions: A Review.

Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the obtainment, identification, and analysis of genetic material. In this context, methods, such as Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), real-time PCR, loopmediated isothermal amplification (LAMP), and loop-mediated isothermal amplification with reverse transcription (RT-LAMP), can be cited. Such methods use enzymes, buffers, and thermosensitive reagents, which require specific storage conditions. In an attempt to solve this problem, the lyophilization procedure (dehydration process by sublimation) can be applied, aiming to preserve and prolong the useful life of the reaction components in cases of temperature variation. In this review, we present a synthesis of the lyophilization process, describing the events of each step of the procedure and providing general information about the technique. Moreover, we selected lyophilization protocols found in the literature, paying attention to the conditions chosen by the authors for each step of the procedure, and structured the main data in tables, facilitating access to information for researchers who need material to produce new functional protocols.

One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

Convective polymerase chain reaction in standard microtubes.

Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed. One of the relevant goals is to shorten PCR duration, which can be achieved in several ways. Here, we report on the results regarding nucleic acids amplification by convective PCR (cPCR) in standard 0.2 ml polypropylene microtubes. The following conditions were found to be optimal for such amplification: 1) 70 µl reaction volume, 2) the supply of external temperature 145°Ð¡ for the denaturation zone and 0°Ð¡ for the annealing zone, 3) ∼30° inclination of the microtube main axis, 4) the use of nearby primers, and 5) duration of the reaction 15-20 min. At these conditions, the amplification products are accumulated in an amount sufficient to be registered by gel electrophoresis, and high sensitivity of the reaction comparable to that of conventional PCR is achieved. cPCR provided the reliable detection of SARS-CoV-2 coronavirus RNA isolated from nasopharyngeal swabs of COVID-19 patients.

An Insight Into Detection Pathways/Biosensors of Highly Infectious Coronaviruses.

The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.

Ocorrência de Babesia sp, Ehrlichia canis e Hepatozoon canis em cães domiciliados, em dois municípios do estado do Espírito Santo - Brasil / Occurrence of Babesia sp, Ehrlichia canis and Hepatozoon canis in domiciled dogs in two municipalities of the state of Espírito Santo - Brazil / Ocurrencia de Babesia sp, Ehrlichia canis y Hepatozoon canis em perros domiciliados em dos municipios del estado de Espírito Santo - Brasil

As doenças transmitidas por carrapatos são afecções de grande importância na clínica médica de pequenos animais, devido à alta casuística e ampla distribuição vetorial no território brasileiro. Os principais agentes responsáveis pelas infecções em cães são Babesia sp., Ehrlichia canis e Hepatozoon canis. Os animais infectados são assintomáticos ou apresentam sinais clínicos inespecíficos, sendo necessário a utilização de testes diagnósticos para definição do agente etiológico, e diagnóstico seguro. O objetivo do presente estudo foi determinar a ocorrência desses micro-organismos em cães naturalmente infectados, domiciliados nos municípios de Vila Velha e Anchieta, Espírito Santo, utilizando diferentes testes de detecção: Reação em cadeia polimerase (PCR), sorologia para detecção de anticorpos anti Ehrlichia canis e pesquisa de hematozoários em esfregaço sanguíneo. Foram analisadas 65 amostras de sangue obtidas por venopunção de veia cefálica de cães. No teste de PCR, 4,62% dos animais foram positivos para Babesia vogeli e 1,54% para Ehrlichia canis sendo os resultados para Hepatozoon canis negativos. No teste sorológico para E. canis 90,77% dos animais foram positivos para a presença de anticorpos, e na pesquisa em lâminas de esfregaço sanguíneo 3,02% apresentavam outros hemoparasitas. Os resultados indicam a dispersão desses hemoparasitas na população canina da região de estudo, entretanto com baixa ocorrência. O teste de PCR demonstrou-se como o mais sensível no qual Babesia vogeli foi o agente mais observado.(AU)

Tick-borne diseases are diseases of great importance in the medical practice of small animals, due to the high casuistry and wide vectorial distribution in the Brazilian territory. The main agents responsible for infections in dogs are Babesia sp., Ehrlichia canis and Hepatozoon canis. Infected animals are asymptomatic or present nonspecific clinical signs, requiring the use of diagnostic tests to define the etiologic agent, and safe diagnosis. The objective of the present study was to determine the occurrence of these microorganisms in naturally infected dogs domiciled in the municipalities of Vila Velha and Anchieta, Espírito Santo, using different detection tests: polymerase chain reaction (PCR), serology to detect antibodies against Ehrlichia canis and research of hematozoa in blood smears. Sixty-five blood samples obtained by venipuncture of the cephalic vein of dogs were analyzed. In the PCR test, 4.62% of the animals were positive for Babesia vogeli and 1.54% for Ehrlichia canis, and the results for Hepatozoon canis were negative. In the serological test for E. canis, 90.77% of the animals were positive for the presence of antibodies, and in the research in blood smear slides, 3.02% presented other hemoparasites. The results indicate the dispersion of these hemoparasites in the canine population of the study region, however with low occurrence. The PCR test proved to be the most sensitive, in which Babesia vogeli was the most observed agent.(AU)

Las enfermedades transmitidas por garrapatas son enfermedades de gran importancia en la práctica médica de los pequeños animales, debido a la alta casuística y amplia distribución vectorial en el territorio brasileño. Los principales agentes responsables de las infecciones en los perros son Babesia sp., Ehrlichia canis y Hepatozoon canis. Los animales infectados son asintomáticos o presentan signos clínicos inespecíficos, siendo necesario el uso de pruebas diagnósticas para la definición del agente etiológico, y el diagnóstico seguro. El objetivo del presente estudio fue determinar la ocurrencia de estos microorganismos en perros infectados naturalmente, domiciliados en los municipios de Vila Velha y Anchieta, Espírito Santo, utilizando diferentes pruebas de detección: reacción en cadena de la polimerasa (PCR), serología para detectar anticuerpos anti Ehrlichia canis e investigación de hematozoos en frotis de sangre. Se analizaron sesenta y cinco muestras de sangre obtenidas por venopunción de la vena cefálica de los perros. En la prueba PCR, el 4,62% de los animales fueron positivos para Babesia vogeli y el 1,54% para Ehrlichia canis, y los resultados para Hepatozoon canis fueron negativos. En la prueba serológica para E. canis, el 90,77% de los animales fueron positivos a la presencia de anticuerpos, y en la investigación en láminas de frotis de sangre el 3,02% presentaron otros hemoparásitos. Los resultados indican la dispersión de estos hemoparásitos en la población canina de la región de estudio, aunque con una baja presencia. La prueba PCR resultó ser la más sensible, en la que Babesia vogeli fue el agente más observado.(AU)

Highly multiplexed rapid DNA detection with single-nucleotide specificity via convective PCR in a portable device.

Assays for the molecular detection of nucleic acids are typically constrained by the level of multiplexing (this is the case for the quantitative polymerase chain reaction (qPCR) and for isothermal amplification), turnaround times (as with microarrays and next-generation sequencing), quantification accuracy (isothermal amplification, microarrays and nanopore sequencing) or specificity for single-nucleotide differences (microarrays and nanopore sequencing). Here we show that a portable and battery-powered PCR assay performed in a toroidal convection chamber housing a microarray of fluorescently quenched oligonucleotide probes allows for the rapid and sensitive quantification of multiple DNA targets with single-nucleotide discrimination. The assay offers a limit of detection of 10 DNA copies within 30 min of turnaround time and a dynamic range spanning 4 orders of magnitude of DNA concentration, and we show its performance by detecting 20 genomic loci and 30 single-nucleotide polymorphisms in human genomic DNA samples, and 15 bacterial species in clinical isolates. Portable devices for the fast and highly multiplexed detection of nucleic acids may offer advantages in point-of-care diagnostics.

Point-of-Care PCR Assays for COVID-19 Detection.

Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR) and the quantitative variant (qRT-PCR) have been the gold standard for COVID-19 diagnosis. However, faster antigen tests and other point-of-care (POC) devices have also played a significant role in containing the spread of SARS-CoV-2 by facilitating mass screening and delivering results in less time. Thus, despite the higher sensitivity and specificity of the RT-PCR assays, the impact of POC tests cannot be ignored. As a consequence, there has been an increased interest in the development of miniaturized, high-throughput, and automated PCR systems, many of which can be used at point-of-care. This review summarizes the recent advances in the development of miniaturized PCR systems with an emphasis on COVID-19 detection. The distinct features of digital PCR and electrochemical PCR are detailed along with the challenges. The potential of CRISPR/Cas technology for POC diagnostics is also highlighted. Commercial RT-PCR POC systems approved by various agencies for COVID-19 detection are discussed.

An updated RT-qPCR assay for the simultaneous detection and quantification of chikungunya, dengue and zika viruses.

The real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for the detection and quantification of arboviruses, including Chikungunya, Dengue, and Zika viruses. In this study, an updated real-time RT-qPCR assay was designed and evaluated together with a synthetic positive-control chimeric RNA for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses. Amplification assays were performed to verify the construct integrity and optimal reaction/thermal cycling conditions. The analytical sensitivity of the assay was determined for each virus in single and multiplex reactions, as well as the performance in the detection and viral load quantification of experimental samples. The real-time RT-qPCR assay presented here allowed for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses and could be applied in several studies where the accurate quantification of viral genomes is required.

Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR.

BACKGROUND: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs. METHODS: A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay. RESULTS: There were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated. CONCLUSIONS: The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.

Pulse-Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions.

BACKGROUND: Molecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity. METHODS/PRINCIPLE FINDINGS: Here we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis. CONCLUSIONS/SIGNIFICANCE: PCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests.

Rapid detection assay of toxigenic Clostridioides difficile through PathOC RightGene, a novel high-speed polymerase chain reaction device.

Nucleic acid amplification tests for diagnosing Clostridioides difficile infections (CDI) are improving to become faster and more accurate. This study aimed to evaluate the accuracy of rapid detection of toxigenic C. difficile using the novel high-speed polymerase chain reaction (PCR) device, PathOC RightGene. These results were compared and evaluated with real-time PCR (qPCR) and enzyme immunoassays (EIA) kit. For this study, 102 C. difficile and 3 Clostridium species isolated from CDI patients were used. These C. difficile isolates were 85 toxigenic and 17 non-toxigenic strains. The results of qPCR served as a standard, and sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the PathOC Right Gene were 99.2%, 99.4%, 100%, 98.8%, and 99.3%, respectively. Turnaround time of qPCR and EIA was 85 and 30 minutes, whereas PathOC RightGene was only 25 minutes including DNA extraction. This novel high-speed PCR device detected toxigenic C. difficile rapidly and accurately.

Effect of Leptin, Pituitary Transcription Factor and Luteinizing Hormone Receptor Genes Polymorphisms on Reproductive Traits and Milk Yield in Holstein Cattle

Abstract The aim of this study was to estimate allelic and genotypic frequencies of markers in the leptin (LEP), pituitary transcription factor (PIT-1) and luteinizing hormone receptor (LHR) genes and evaluate their effects on reproductive traits and milk yield of Holstein cattle. Data from 147 cows from department of Francisco Morazán, Honduras, were collected and PCR-Restriction Fragment Length Polymorphism (RFLP) assays were performed to characterize the PIT-1-HinfI, LEP- A59V and LHR-rs41256848 polymorphisms. To estimate the effect of genotypes on reproductive traits and milk yield fixed and mixed linear models were fitted. The frequencies of the genotypes CC, CT and TT of A59V, AA, AB and BB of HinfI, and CC, CG and GG of rs41256848 were 0.46, 0.33 and, 0.21; 0.09, 0.32 and 0.58; and 0.37, 0.61 and 0.02, respectively. The genotypes of LEP and LHR showed deviations from Hardy-Weinberg equilibrium. The A59V polymorphism was significantly associated with the calving to conception interval (CCI) (p=0.01), being the C allele favorable. The HinfI and rs41256848 polymorphism were significantly associated (p=0.08 and p=0.04) with age to first calving (AFC), being the A and G the alleles favorable associated, respectively. The results suggest that LEP, PIT and LHR polymorphisms can probably act as candidate to be used in marker-assisted selection for AFC and CCI traits.

High-throughput and uniform large field-of-view multichannel fluorescence microscopy with super-thin dichroism for a dPCR gene chip.

With the rapid development of digital precision medicine, the digital polymerase chain reaction (dPCR) deoxyribonucleic acid (DNA) gene chip integrates more channels with smaller size and larger area, which leads to a higher technical requirement for commercial optical fluorescence microscopy. The multitime image splicing method is widely used for DNA detection. However, it consumes time and has visible seamless image results. This work has demonstrated the design and fabrication of a three channel reversed and reduced fluorescence microscopic imaging system with high-resolution and large field of view for one-time imaging. We introduced the super ultra-thin dichroic mirror into the space between the objective lens and the gene chip to achieve a uniform illumination and a strong signal for the large area gene chip. The fabricated new fluorescence microscopy can take a one-time imaging for the 28×18mm dPCR gene chip with more than 20,000 multi micro-droplets within FAM, HEX, and ROX fluorescence channels. The optical system was designed with a numerical aperture (NA) of 0.106. Modulation transfer function (MTF) is higher than 0.675 at 70 lp/mm, and the function resolution capability is 10 µm with the whole magnification of -0.65times. The fly's eye lens-based illumination system was tested with a uniform output of over 90% in the whole ϕ34.7mm chip area. The design was tested, and the experimental results showed that this new system provides a fast, efficient, and professional optical imaging method for detection of the new emerged digital PCR gene chip, which has larger area and more channels.

Single Extracellular Vesicle Protein Analysis Using Immuno-Droplet Digital Polymerase Chain Reaction Amplification.

There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno-droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody-DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read-out by droplet imaging. In these proof-of-principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.

Estimation of the sensitivity and specificity of four serum ELISA and one fecal PCR for diagnosis of paratuberculosis in adult dairy cattle in New Zealand using Bayesian latent class analysis.

In New Zealand, a new diagnostic approach for the control of paratuberculosis in mixed aged milking cows has been developed using a combination of ELISA and quantitative fecal PCR (f-qPCR). Our analysis was designed to evaluate performance of these individual tests in infected or infectious mixed aged cows across the prevalence of infection typically encountered on NZ dairy farms and calculate test accuracy when used as a screening test of serological ELISAs for four separate antigens read in parallel followed by a confirmatory quantitative f-qPCR test. Data from a cross-sectional study of 20 moderate prevalence herds was combined with existing data from 2 low and 20 high prevalence herds forming a dataset of 3845 paired serum and fecal samples. Incidence of clinical Johne's disease (JD) was used to classify herds into three prevalence categories. High (≥ 3% annual clinical JD for the last three years), moderate (<3 - 1%) and low (<1% incidence for at least the last five years). Positive tests were declared if> 50 ELISA units and f-qPCR at two cut-points (≥1 × 104 genomes/mL or >1 × 103 genomes/mL). Fixed Bayesian latent class models at both f-qPCR cut-points, accounted for conditional independence and paired conditional dependence. Mixed models at both f-qPCR cut-points, using a different mechanism to account for conditional dependencies between tests were also implemented. Models (24 in number) were constructed using OpenBUGS. The aim was to identify Mycobacterium avium subsp. paratuberculosis (MAP) infected cows that met at least one of two criteria: shedding sufficient MAP in feces to be detected by f-qPCR or mounting a detectable MAP antibody response. The best fit to the data was obtained by modelling pairwise dependencies between tests in a fixed model or by accounting for dependencies in a mixed model at a fecal cut-off of ≥1 × 104 genomes/mL. Test performance differed with prevalence, but models were robust to prior assumptions. For the fixed model, at a prevalence of 0.29 (95 % probability interval (PI) = 0.25-0.33), as a screening plus confirmatory f-qPCR, post-test probability for disease in a positive animal was 0.84 (95 %PI = 0.80-0.88) and 0.16 (95 %PI = 0.15-0.18) for disease in a test negative animal. In low prevalence herds (0.01(95 %PI = 0.00-0.04)) the equivalent figures were 0.84 (95 %PI = 0.08-0.92) and 0.00 (95 %PI = 0.00-0.02). These results suggest this is a useful tool to control JD on dairy farms, particularly in herds with higher levels of infection, where the sampling and testing cost per animal is defrayed across more detected animals.

A digital PCR system based on the thermal cycled chip with multi helix winding capillary.

This paper presents a digital PCR system based on a novel thermal cycled chip, which wraps microchannels on a trapezoidal structure made of polydimethylsiloxane (PDMS) in a multi-helix manner for the first time. It is found that compared to the single helix chip commonly used in previous reports, this kind of novel multi-helix chip can make the surface temperature in the renaturation zone more uniform, and even in the case of rapid fluid flow, it can improve the efficiency of the polymerase chain reaction. What's more, the winding method of multi helix (such as double helix, six helix and eight helix) can obtain better temperature uniformity than the winding of odd helix (such as single helix and three helix). As a proof of concept, the temperature-optimized double-helical chip structure is applied to continuous-flow digital PCR and there is no need to add any surfactant to both the oil phase and reagent. In addition, we successfully analyzed the fluorescence signal of continuous-flow digital PCR by using CMOS camera. Finally, this method is applied for the absolute quantification of the clinical serum sample infected by HBV. The accuracy of the test results has been confirmed by commercial instruments.

Diretor do Lacen/MA, Luiz Fernando Ferreira fala sobre Diagnóstico Laboratorial para COVID-19

O diretor do Lacen e secretário geral do Conselho Regional de Farmácia do Maranhão, Luiz Fernando Ramos Ferreira, participou Debate Virtual do Conass, no dia 31/07, que abordou os testes da Covid-19 – quais os tipos, quando e quais devem ser usados, como ser aplicados, como fazer a leitura do resultado, entre outros aspectos relevantes deste instrumento fundamental para o enfrentamento e controle da pandemia do novo coronavírus. A iniciativa faz parte da implantação do Guia Orientador do Enfrentamento da Covid-19 na Rede de Atenção à Saúde, elaborado em parceria com o Conselho Nacional de Secretarias Municipais de Saúde (Conasems). Também participaram o assessor técnico do Conass, Fernando Avendanho; o infectologista, gestor hospitalar e médico de Genotipagen do HIV, Boaventura Queiroz; o assessor técnico do Conass, Nereu Henrique Mansano; e o assessor técnico do Conasems, Alessandro Chagas. A mediação do debate será feita pela assessora técnica do Conass, Maria José Evangelista.