RT-PCR para identificação de mutação do receptor do fator de crescimento epidérmico (EGFR) em pacientes com câncer de pulmão de células não pequenas / RT-PCR for identification of epidermal growth factor receptor (EGFR) mutation in patients with non-small cell lung cancer

INTRODUÇÃO: O câncer de pulmão foi o segundo tipo de câncer mais diagnosticado (11,4%) no mundo e a principal causa de morte por câncer (18%) durante o ano de 2020, totalizando 2,2 milhões de novos casos e 1,8 milhões de mortes no ano. Considera-se para fins terapêuticos e prognósticos a divisão em dois principais tipos histológicos: câncer de pulmão células não pequenas células (CPCNP), que representa cerca de 85% de todos os casos de câncer de pulmão, e câncer de pulmão pequenas células (CPPC), associado a aproximadamente 15% dos casos. O comportamento biológico dos tumores malignos pode estar relacionado à expressão molecular, principalmente de proteínas envolvidas no crescimento e sobrevivência celulares, de modo que a segurança e a eficácia do tratamento guardam relação não só com o subtipo histopatológico como também com as características moleculares do tumor. Assim, para melhor direcionamento da terapia (por exemplo, erlotinibe e gefinitibe), torna-se importante diferenciar os subtipos histológicos, e identificar a presença de mutações específicas, como, por exemplo formas alteradas no gene do EGFR, como se recomenda nas Diretrizes Diagnósticas e Terapêuticas do Câncer de Pulmão do Ministério da Saúde. Entretanto, o Ministério da Saúde não recomenda nenhuma técnica específica para identificação de mutação no gene do EGFR, sendo que o rt-PCR é considerado o teste de referência em muitos contextos clínicos e estudos, seguido pelo sequenciamento de nova geração (NGS). PERGUNTA: Em pacientes diagnosticados com CPCNP, qual a acurácia diagnóstica, custoefetividade e impacto orçamentário do diagnóstico molecular por meio da técnica de rtPCR para identificação de mutação do EGFR em comparação ao NGS ou a não realização de rt-PCR? EVIDÊNCIAS CIENTÍFICAS: A revisão sistemática identificou 19 estudos observacionais de acurácia diagnóstica (teste de referência: NGS). A sensibilidade e especificidade do rt-PCR em comparação ao NGS foi de 92,0% e 97,0%, respectivamente. Apenas um estudo reportou dados de sobrevida global para a comparação de rt-PCR positivo e negativo para mutação de EGFR, apresentando uma diferença não significativa estatisticamente, de 19,2 versus 11,6 meses (Log rank p=0,143). Todos os estudos apresentaram ao menos um domínio com alto risco de viés e nenhuma preocupação quanto à aplicabilidade. AVALIAÇÃO ECONÔMICA: Na análise de custo-efetividade/utilidade comparou-se a realização de rt-PCR com a não realização do teste para um horizonte temporal por toda vida. A análise sugere que a realização do rt-PCR de mutação do EGFR para auxiliar na padronização do melhor tratamento de pacientes com CPCNP e sem tratamento prévio na fase metastática, em comparação a não realização do teste, sugere um modesto benefício clínico de 0,043 ano de vida ajustado pela qualidade (QALY) e 0,040 ano de vida (AV) ganho e uma economia de R$ 3.138,25. Portanto, rt-PCR dominou a não realização do teste (razão de custo-efetividade incremental, RCEI, não representada em casos de dominância). Ademais, as análises de sensibilidade determinística revelaram que para QALY e AV, as proporções de pacientes que usam o erlotinibe e gefitinibe são as variáveis que mais impactam na RCEI, seguida pela proporção de resultados positivos obtidos pela rt-PCR de mutação do EGFR. AVALIAÇÃO DE IMPACTO ORÇAMENTÁRIO: Considerando um horizonte temporal de cinco anos, utilizando demanda aferida (dados de APAC de 2015 a 2021) foi estimado o quantitativo de pacientes com carcinoma pulmonar de células não pequenas avançado. Ademais, foi estimada uma taxa de difusão conservadora, tendo um aumento de 10% ao ano para a tecnologia em análise. Desta forma, observou-se que a incorporação do rt-PCR para identificação de mutação do EGFR em pacientes com CPCNP, sem tratamento prévio na fase metastática, pode gerar um incremento de R$ 1.553.250,00 no primeiro ano, chegando a um incremento de R$ 8.193.750,00 no quinto ano de análise (total acumulado de cerca de R$ 24 milhões em cinco anos) em análise que não considera as potenciais economias com a incorporação do teste. Em análise de sensibilidade considerando preço 20% menor, coerente com preço informado por um dos fabricantes contatados, o impacto orçamentário acumulado em cinco anos seria de cerca de R$ 20 milhões. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: No Monitoramento do Horizonte Tecnológico foi encontrada 1 tecnologia, a Allele-Discriminating Priming System (kit - ADPS), da empresa Genecast, sem identificação de licenças sanitárias Anvisa e FDA. CONSIDERAÇÕES FINAIS: A evidência disponível sobre o uso de rt-PCR para identificação de mutação EGFR em comparação ao NGS sugere elevada acurácia do rt-PCR, com sensibilidade e especificidade de 92,0% e 97,0%, respectivamente. A análise de custoefetividade sugere que a realização da rt-PCR de mutação do EGFR em pacientes com CPCNP, em comparação a não realização do teste, é dominante para QALY e AV. A análise de impacto orçamentário da incorporação da rt-PCR de mutação do EGFR em pacientes com CPCNP, no SUS, sugere um incremento de cerca de R$ 21 a 40 milhões em cinco anos de análise, a depender do preço do kit e market share considerado, em análise que não considera a potencial economia com a incorporação do teste. RECOMENDAÇÃO PRELIMINAR DA CONITEC: Os membros do Comitê de Procedimentos e Produtos, presentes na 123ª Reunião Ordinária da Conitec realizada no dia 05 de outubro de 2023, deliberaram por unanimidade que a matéria fosse disponibilizada em consulta pública com recomendação preliminar favorável à incorporação do diagnóstico molecular por meio da técnica de rt-PCR para identificação de mutação do EGFR em pacientes com câncer de pulmão de células não pequenas. CONSULTA PÚBLICA: A consulta pública nº 51 foi realizada entre os dias 12 de dezembro de 2023 e 02 de janeiro de 2024. Foram recebidas 50 contribuições, sendo 6 classificadas como técnico-científicas e 44 como de experiência ou opinião. Todas as contribuições foram favoráveis à incorporação de rt-PCR para identificação de mutação do EGFR em pacientes com CPCNP. Referente às contribuições técnico-científicas, os estudos sugeridos para inclusão no PTC não contemplavam os critérios de elegibilidade da pergunta PIROS e todas as demais contribuições corroboram os resultados apresentados no relatório de recomendação. Quanto às contribuições de experiência e opinião, das 44 recebidas, uma foi desconsiderada por tratar de outra consulta pública. Também foram recebidos dois anexos. As contribuições recebidas e anexos foram submetidos à análise de conteúdo temática. Das 43 contribuições consideradas, todas expressaram concordância em relação à recomendação preliminar da Conitec, portanto, mostraramse favoráveis à incorporação do procedimento em avaliação. Os participantes que tiveram experiência com o rt-PCR relataram como efeitos positivos a importância do uso e acesso ao teste para os pacientes usuários do SUS terem um melhor direcionamento no tratamento do câncer de pulmão de células não pequenas. Enquanto efeitos negativos e dificuldades no uso do rt-PCR, foram apontados a dificuldade no acesso e o custo. RECOMENDAÇÃO FINAL DA CONITEC: Os membros do Comitê de Produtos e Procedimentos presentes na 126ª Reunião Ordinária da Conitec deliberaram, por unanimidade, recomendar a incorporação de rt-PCR para identificação da mutação do receptor do fator de crescimento epidérmico (EGFR) em pacientes com câncer de pulmão de células não pequenas. Foi assinado o Registro de Deliberação nº 876/2024. DECISÃO: e incorporar, no âmbito do Sistema Único de Saúde - SUS, o RT-PCR para identificação de mutação do receptor do fator de crescimento epidérmico (EGFR) em pacientes com câncer de pulmão de células não pequenas, publicada no Diário Oficial da União nº 46, seção 1, página 54, em 07 de março de 2024.

Reacción cadena polimerasa en tiempo real para detección del virus papiloma humano (VPH) cáncer de cérvix / Real-time polymerase chain reaction for detection of human papillomavirus (HPV) cervical cancer

INTRODUCCIÓN: El cáncer de cérvix ha ocupa el primer lugar en las neoplasias con mayor número de casos nuevos registrados en el INEN durante los últimos 10 años. - El virus del papiloma humano (VPH) es fundamental para el desarrollo de neoplasias de cuello uterino y puede detectarse en el 99,7% de los cánceres de cuello uterino. - PCR y captura de híbridos (CH2) son dos técnicas aprobadas nacional y mundialmente para el diagnóstico de VPH. - El diagnóstico por PCR tiene la ventaja de diferenciar oncogenes de alto grado, especialmente 16 y 18 para VPH, en comparación con CH2. METODOLOGÍA: Se revisó literatura existente respecto a la técnica de diagnóstico por PCR para VPH. Se encontró una revisión sistemática y tres estudios clínicos primario. DISCUSIÓN: Ambas pruebas tienen una concordancia mayor del 80%, sin embargo, la prueba PCR diferencia los subtipos oncogénicos y no oncogénicos. - La evidencia es discordante, respecto a la sensibilidad y especificad de las pruebas de PCR y CH2, pues estas varían según la prevalencia de la enfermedad y la población evaluada. Sin embargo, muestra mayor especificidad para PCR. - En el Perú se realizan detección de VPH con PCR-TR. Las agencias reguladoras como la FDA han aprobado el uso de esta tecnología en diferentes plataformas de diagnóstico, así como el MINSA aprobado el uso de diagnóstico de VPH con técnicas moleculares. - Para la implementación de esta nueva tecnología se debe contar con los reactivos, profesional médico capacitado para la lectura de los resultados y contar con el personal médico que atienda a quienes den positivo a la prueba de VPH. - Se sugiere el uso de PCR-TR para el diagnóstico de VPH en el INEN. CONCLUSIONES: El virus del papiloma humano (VPH) es fundamental para el desarrollo de neoplasias de cuello uterino y puede detectarse en el 99,7% de los cánceres de cuello uterino. PCR y captura de híbridos (CHII) son dos técnicas aprobadas nacional y mundialmente para el diagnóstico de VPH. El diagnóstico por PCR tiene la ventaja de diferenciar oncogenes de alto grado, especialmente 16 y 18 para VPH, en comparación con CHII. Se revisó literatura existente respecto a la técnica de diagnóstico por PCR para VPH. Se encontró una revisión sistemática y algunos estudios clínicos primario. Ambas pruebas tienen una concordancia mayor del 80%, sin embargo, la prueba PCR diferencia los subtipos oncogénicos y no oncogénicos. La evidencia es discordante, respecto a la sensibilidad y especificad de las pruebas de PCR y CHII. Sin embargo, muestra mayor especificidad para PCR.

Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study.

We sought to develop a smooth and low cost sample preparation and DNA extraction protocol, streamlined with a ready-to-use qPCR in a portable instrument to overcome some of the existing hurdles. Several solutions were evaluated as to their ability to liquefy a mucin-based matrix. Each liquefied matrix, supplemented with either Mycobacterium tuberculosis (MTB) H37Rv strain DNA or intact cells, was aliquoted onto a filter paper embedded with solubilizing agents, and was subsequently dried up. Most of the nucleic acids, including genomic DNA from the bacilli and the host, binds to the filter paper. Next, several protocols were evaluated to elute the DNA from the paper, using qPCR to detect the insertion sequence IS6110, a M. tuberculosis complex genomic marker. The limit of detection (LOD) of the best protocol was then evaluated using parallel seeding and colony counting. The protocol was also evaluated using seventeen sputum samples, previously characterized by the GeneXpert or culture. Two instruments (the ABI7500 Standard and the Q3-Plus system) and two reagents storage formats (frozen or ready-to-use) were evaluated. Solutions containing guanidine isothiocyanate exerted the best liquefying effect on the mucin-based matrix extracted from one 6-mm punches, followed by a brief incubation at 95°C. The resulting DNA contained impurities, but a simple 1:10 dilution elicited the detection of MTB and human genomic targets. The described protocol presented an apparent LOD of 02 CFU/mL of MTB. Challenging the protocol with previously characterized samples showed substantial agreement with GeneXpert MTB/RIF results (sensitivity of 90%, agreement of 88.9%, kappa coefficient of 0.77), and moderate agreement with culture results (sensitivity of 100%, agreement of 78.9%, kappa coefficient of 0.58). This work presents a sensitive proof-of-concept protocol for sputum liquefaction and decontamination followed by a simple DNA extraction procedure, in which the extraction steps are streamlined with a ready-to-use qPCR in a portable instrument that can be employed in low infrastructure settings.

Rapid detection of novel coronavirus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification.

Novel Corona virus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.

High-Throughput Single-Cell Real-Time Quantitative PCR Analysis.

Examining transcriptomics of populations at the single-cell level allows for higher resolution when studying functionality in development, differentiation, and physiology. Real-time quantitative PCR (qPCR) enables a sensitive detection of specific gene expression; however, processing a large number of samples for single-cell research involves a time-consuming process and high reagent costs. Here we describe a protocol for single-cell qPCR using nanofluidic chips. This method reduces the number of handling steps and volumes per reaction, allowing for more samples and genes to be measured.

Two-tailed RT-qPCR panel for quality control of circulating microRNA studies.

Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).

Cost-effectiveness of GeneXpert and LED-FM for diagnosis of pulmonary tuberculosis: A systematic review.

BACKGROUND: Early and accurate diagnosis of tuberculosis is a priority for TB programs globally to initiate treatment early and improve treatment outcomes. Currently, Ziehl-Neelsen (ZN) stain-based microscopy, GeneXpert and Light Emitting Diode-Fluorescence Microscopy (LED-FM) are used for diagnosing pulmonary drug sensitive tuberculosis. Published evidence synthesising the cost-effectiveness of these diagnostic tools is scarce. METHODOLOGY: PubMed, EMBASE and Cost-effectiveness analysis registry were searched for studies that reported on the cost-effectiveness of GeneXpert and LED-FM, compared to ZN microscopy for diagnosing pulmonary TB. Risk of bias was assessed independently by four authors using the Consensus Health Economic Criteria (CHEC) extended checklist. The data variables included the study settings, population, type of intervention, type of comparator, year of study, duration of study, type of study design, costs for the test and the comparator and effectiveness indicators. Incremental cost-effectiveness ratio (ICER) was used for assessing the relative cost-effectiveness in this review. RESULTS: Of the 496 studies identified by the search, thirteen studies were included after removing duplicates and studies that did not fulfil inclusion criteria. Four studies compared LED-FM with ZN and nine studies compared GeneXpert with ZN. Three studies used patient cohorts and eight were modelling studies with hypothetical cohorts used to evaluate cost-effectiveness. All these studies were conducted from a health system perspective, with four studies utilising cost utility analysis. There were considerable variations in costing parameters and effectiveness indicators that precluded meta-analysis. The key findings from the included studies suggest that LED-FM and GeneXpert may be cost effective for pulmonary TB diagnosis from a health system perspective. CONCLUSION: Our review identifies a consistent trend of the cost effectiveness of LED-FM and GeneXpert for pulmonary TB diagnosis in different countries with diverse context of socio-economic condition, HIV burden and geographical distribution. However, all the studies used different parameters to estimate the impact of these tools and this underscores the need for improving the methodological issues related to the conduct and reporting of cost-effectiveness studies.

Variability and cost implications of three generations of the Roche LightCycler® 480.

Real time PCR has become a dominant method for the highly sensitive detection of pathogens in clinical material. Real time PCR can generate a fluorescence signal by using fluorescence labelled probes, allowing us to detect and semi quantify the amount of amplified DNA. Here we test the variability of the detection system and cost implications of three different versions of the LightCycler® 480 (LC480), focusing on the intensity of fluorescence and Cq in monoplex and multiplex rtPCRs. For gastro-intestinal pathogens there was no correlation between the intensity of fluorescence and the Cq value in the different LC480 types. For probes with the dyes FAMTM, HEXTM, Cy5 and Red610 a higher fluorescence intensity was seen in LC480 type II and III compared to LC480 type I. After lowering the probe concentration for the Cy5 dye three-fold (from 0.3µM to 0.1µM) the Cq value remains the same and the intensity of fluorescence decreases. For the LC480 type II and III the difference in fluorescence intensity was much more extreme. The concentration of the different labelled probes can be lowered at least six-fold in LC480 type II and III cyclers while maintaining a fluorescence intensity as high as achieved in the LC480 type I with undiluted probe. In conclusion, the strength of the fluorescence signal of the LightCycler® 480 type III is superior to that of LightCycler® 480 types I and II, allowing the use of lower probe concentrations for all dyes, particularly for the dyes Red610 and Cy5. This results in a two thirds reduction in PCR probe costs. Switching to these newer machines for real-time PCR can reduce dye labelled probe consumption and thus reduce costs significantly.

Development and validation of InnoQuant® HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets.

The development and validation of InnoQuant® HY, a real-time PCR system containing four DNA targets-two RE autosomal targets of different sizes, male specific targets, and an internal positive control target-are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition. The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow can be created that minimizes unnecessary sample processing and retesting while maximizing recovery of probative DNA profiles from challenging biological evidence.

Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients.

BACKGROUND: Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. OBJECTIVES: First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. STUDY DESIGN: We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. RESULTS: The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p<0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p>0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2h for DFA, 31.8€ and 1.5h for Simplexa™ and 55€ and 3h for TAC testing. CONCLUSIONS: Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.

Evaluation of a dilution method for non-evaluable results in the detection of Chlamydia trachomatis and Neisseria gonorrhoeae with the Cobas 4800 platform. / Valoración de un método de dilución para la resolución de resultados no valorables en la detección de Chlamydia trachomatis y Neisseria gonorrhoeae con la plataforma cobas 4800.

INTRODUCTION: A variable percentage of samples analysed using the Cobas 4800 assay can give an invalid result by PCR inhibition or erroneous due to incorrect DNA extraction with the Cobas 4800 CT/NG test. METHOD: An analysis was performed using the vortex agitation and dilution protocol on the original sample (swab or urine) for a total of 116 samples. In order to analyse the sensitivity of this method, 100 samples (swabs and urine) with known results were retested. RESULTS: A total of 98.3% (114/116) of the samples analysed were resolved with this protocol with 100% agreement after reviewing clinical data, Gram stain, and other samples analysed in parallel from the same patient. DISCUSSION: The data indicate no loss of sensitivity with this protocol; thus Cobas 4800 users could use this method without the need for alternative methods.

A Low-Cost and Fast Real-Time PCR System Based on Capillary Convection.

A low-cost and fast real-time PCR system in a pseudo-isothermal manner with disposable capillary tubes based on thermal convection for point-of-care diagnostics is developed and tested. Once stable temperature gradient along the capillary tube has been established, a continuous circulatory flow or thermal convection inside the capillary tube will repeatedly transport PCR reagents through temperature zones associated with the DNA denaturing, annealing, and extension stages of the reaction. To establish stable temperature gradient along the capillary tube, a dual-temperature heating strategy with top and bottom heaters is adopted here. A thermal waveguide is adopted for precise maintenance of the temperature of the top heater. An optimized optical network is developed for monitoring up to eight amplification units for real-time fluorescence detection. The system performance was demonstrated with repeatable detection of influenza A (H1N1) virus nucleic acid targets with a limit of detection of 1.0 TCID50/mL within 30 min.

Evaluation of a Commercial Real-Time PCR Kit for the Detection of Mycobacterium avium subspecies paratuberculosis in Milk.

There are several commercial test kits for Mycobacterium avium subspecies paratuberculosis (MAP) detection, each with different advantages, disadvantages, and applications. In the present study, a real-time PCR kit targeting the unique transposon sequence ISMAP02 was evaluated. The analytical sensitivity was determined using the type strain ATCC 19698, and the specificity was validated by testing fifteen MAP isolates, thirteen non-MAP Mycobacterium isolates, and eight non-Mycobacterium isolates. Six spiking experiments were performed using raw milk and reconstituted infant milk artificially contaminated with dilutions containing 10(0)-10(5) MAP cells mL(-1). Sensitivity and specificity were at 100 %. The detection probabilities in raw milk and reconstituted infant milk for the samples (containing 1.4 × 10(1) and 1.7 × 10(1) MAP cell 50 mL(-1)) were 16.6 and 91.6 %, respectively. Thus, the tested kit yielded satisfying results to detect MAP in milk.

Low-cost, real-time, continuous flow PCR system for pathogen detection.

In this paper, we present a portable and low cost point-of-care (POC) PCR system for quantitative detection of pathogens. Our system is based on continuous flow PCR which maintains fixed temperatures zones and pushes the PCR solution between two heated areas allowing for faster heat transfer and as a result, a faster PCR. The PCR system is built around a 46.0 mm × 30.9 mm × 0.4 mm disposable thermoplastic chip. In order to make the single-use chip economically viable, it was manufactured by hot embossing and was designed to be compatible with roll-to-roll embossing for large scale production. The prototype instrumentation surrounding the chip includes two heaters, thermal sensors, and an optical system. The optical system allows for pathogen detection via real time fluorescence measurements. FAM probes were used as fluorescent reporters of the amplicons generated during the PCR. To demonstrate the function of the chip, two infectious bacteria targets were selected: Chlamydia trachomatis and Escherichia coli O157:H7. For both bacteria, the limit of detection of the system was determined, PCR efficiencies were calculated, and different flow velocities were tested. We have demonstrated successful detection for these two bacterial pathogens highlighting the versatility and broad utility of our portable, low-cost, and rapid PCR diagnostic device.

Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.

Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method.

A lab-on-a-chip system integrating tissue sample preparation and multiplex RT-qPCR for gene expression analysis in point-of-care hepatotoxicity assessment.

A truly practical lab-on-a-chip (LOC) system for point-of-care testing (POCT) hepatotoxicity assessment necessitates the embodiment of full-automation, ease-of-use and "sample-in-answer-out" diagnostic capabilities. To date, the reported microfluidic devices for POCT hepatotoxicity assessment remain rudimentary as they largely embody only semi-quantitative or single sample/gene detection capabilities. In this paper, we describe, for the first time, an integrated LOC system that is somewhat close to a practical POCT hepatotoxicity assessment device - it embodies both tissue sample preparation and multiplex real-time RT-PCR. It features semi-automation, is relatively easy to use, and has "sample-in-answer-out" capabilities for multiplex gene expression analysis. Our tissue sample preparation module incorporating both a microhomogenizer and surface-treated paramagnetic microbeads yielded high purity mRNA extracts, considerably better than manual means of extraction. A primer preloading surface treatment procedure and the single-loading inlet on our multiplex real-time RT-PCR module simplify off-chip handling procedures for ease-of-use. To demonstrate the efficacy of our LOC system for POCT hepatotoxicity assessment, we perform a preclinical animal study with the administration of cyclophosphamide, followed by gene expression analysis of two critical protein biomarkers for liver function tests, aspartate transaminase (AST) and alanine transaminase (ALT). Our experimental results depict normalized fold changes of 1.62 and 1.31 for AST and ALT, respectively, illustrating up-regulations in their expression levels and hence validating their selection as critical genes of interest. In short, we illustrate the feasibility of multiplex gene expression analysis in an integrated LOC system as a viable POCT means for hepatotoxicity assessment.

A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification.

Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.

Assessment of DNA Integrity, Applications for Cancer Research.

Many methods have been developed for DNA integrity assessment including electrophoresis-based procedures, quantitative PCR, and, more recently, microfluidics-based procedures. DNA integrity evaluation can be employed for characterizing biological samples quality before extensive genomic analysis and also finds applications in reproductive medicine, prenatal diagnostics, or cancer research. In this chapter, we will focus on the assessment of DNA integrity in cancer research. In particular, we will present the application of the determination of DNA integrity for tracking of circulating tumor DNA. Finally, we will conclude by illustrating the potential innovative application of DNA integrity as a biomarker in clinical research, especially for prognostic purposes, patient follow-up, or early diagnosis.

Quantification and viability assays of Toxoplasma gondii in commercial "Serrano" ham samples using magnetic capture real-time qPCR and bioassay techniques.

"Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. To make Serrano ham, pork undergoes a salting and a subsequent fermentation process known as curing. Certain pigs used for meat production are an important source of Toxoplasma gondii infection in humans. We have developed a method for quantifying and assaying the viability of the T. gondii present in commercial Serrano ham samples. A magnetic capture method for the isolation of T. gondii DNA and a qRT-PCR were used to estimate the T. gondii burden in 475 commercial samples of "Serrano" ham in two presentation formats: ham pieces and sliced ham. The infectivity capacity of T. gondii in positive samples was assayed in mice. The global prevalence of T. gondii was 8.84%, ranging from 32.35% in one of the companies to 0% prevalence in three other companies. The infectivity assays revealed that only 4.84% of the positive samples were infective. To the best of our knowledge this is the first report focussing on the prevalence of T. gondii in commercial "Serrano" ham. The method described here could be useful for producers to guarantee the safety of their products.