Polymerase Chain Reaction Reaction Research Articles

Machine-Free Polymerase Chain Reaction with Triangular Gold and Silver Nanoparticles.

Photothermal effects of metal nanoparticles (NPs) are used for various biotechnological applications. Although NPs have been used in a polymerase chain reaction (PCR), the effects of shape on the photothermal properties and its efficiency on PCR are less explored. The present study reports the synthesis of triangular gold and silver NPs, which can attain temperatures up to ∼90 °C upon irradiation with 808 nm laser. This photothermal property of synthesized nanoparticles was evaluated using various concentrations, irradiation time, and power to create a temperature profile required for variable-temperature PCR. This study reports a cost-effective, machine-free PCR using both gold and silver triangular NPs, with efficiency similar to that of a commercial PCR machine. Interestingly, addition of triangular NPs increases PCR efficiency in commercial PCR reactions. The higher PCR efficiencies are due to the direct binding and unfolding of double-stranded DNA as suggested by circular dichroism and UV spectroscopy. These findings suggest that triangular NPs can be used to develop cost-effective, robust machine-free PCR modules and can be used in various other photothermal applications.

HPV detection in oral mucosa samples in pediatric patients

ABSTRACT INTRODUCTION: The human papillomavirus (HPV) detection favors treatments for patients with clinical manifestations and limits future consequences for those with asymptomatic infections. OBJECTIVES: Therefore, the present study aimed to evaluate the sensitivity of polymerase chain reaction (PCR) for HPV detection from oral mucosa samples, of asymptomatic patients and patients with clinical manifestations of laryngeal papillomatosis. MATERIAL AND METHODS: A total of 49 pediatric patient samples were obtained by exfoliation of the oral mucosa with a sterile brush. The deoxyribonucleic acid (DNA) samples was extracted and used for HPV detection, using GP5 and GP6 oligonucleotides, by conventional PCR and qPCR reactions. RESULTS: Among the 49 samples, eight were from patients clinically diagnosed with laryngeal papillomatosis, but in both conventional PCR and qPCR technic, only one sample had presented positivity. DISCUSSION: These results suggest that the sample type, the methodology used to collect, the extraction methodology used, the anatomical location of the lesion and the oligonucleotides used; all factors strongly influence the sensitivity of HPV detection by PCR methodology. CONCLUSION: Thus, more studies are needed to better determine the sample collection, and the processing techniques present more reproducibility on PCR detection.

A multiplex PCR method for rapid identification of commercially important seaweeds Kappaphycus alvarezii, Kappaphycus striatus and Eucheuma denticulatum (Rhodophyta, Solieriaceae)

Taxonomic clarification of Kappaphycus alvarezii, Kappaphycus striatus and Eucheuma denticulatum based on standard DNA barcodes has proven to be useful as the phenotypic plasticity of the species can confound traditional taxonomic approaches. Molecular characterization of each isolate is time-consuming and a painstaking task as multiple runs of PCR amplification needs to be carried out and the identity of the isolates is only known after sequencing of the PCR amplicons. In this study, we have developed a simple, efficient and reproducible one-step polymerase chain reaction (PCR) based method for the identification of the three eucheumatoid species, K. alvarezii, K. striatus and E. denticulatum. Based on the nuclear ribosomal internal transcribed spacer (ITS) sequence, all the available genetic polymorphisms were reviewed and three primers were designed. The combination of these primers in the same PCR reaction results in a specific amplicon of distinct size ranging from approximately 140-400 bp to be produced for each of the three red algal species. The proposed method is found to be an effective and practical tool to aid in the selection of the desired seaweed seedlings even before they are cultivated and also the identification of commercial cultivars accurately.

Salt-Tolerant Phenomena, Sequencing and Characterization of a Glyoxalase I (Jojo-Gly I) Gene from Jojoba in Comparison with Other Glyoxalase I Genes.

Plant response to salt stress and the mechanism of salt tolerance have received major focus by plant biology researchers. Biotic stresses cause extensive losses in agricultural production globally, but abiotic stress causes significant increase in the methylglyoxal (MG) level of Glyoxalase I (Gly I). Identification of salt-tolerant genes when characterizing their phenotypes will help to identify novel genes using polymerase chain reaction (PCR) to amplify the DNA coding region for glyoxalase I. This method is specific, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. This method was used rapidly and easily identified glyoxalase I sequences as salt-tolerant genes from Jojoba (Simmondsia chinensis (Link) Schneider). In the present study, the glyoxalase I gene was isolated, amplified by PCR using gene-specific primers and sequenced from the jojoba plant, then compared with other glyoxalase I sequences in other plants and glyoxalase I genes like in Brassica napus, ID: KT720495.1; Brassica juncea ID: Y13239.1, Arachis hypogaea; ID: DQ989209.2; and Arabidopsis thaliana L, ID: AAL84986. The structural gene of glyoxalase I, when sequenced and analyzed, revealed that the uninterrupted open reading frame (ORF) of jojoba Gly I (Jojo-Gly I) spans 775 bp, corresponding to 185 amino acid residues, and shares 45.2% amino acid sequence identity to jojoba (Jojo-Gly I). The cloned ORF, in a multicopy constitutive expression plasmid, complemented the Jojo-Gly I, confirming that the encoded Jojo-Gly I in jojoba showed some homology with other known glyoxalase I sequences of plants.

An assumption-free quantitative polymerase chain reaction method with internal standard

In real-time quantitative polymerase chain reaction (PCR), the standard curve between threshold cycle and logarithm of template concentration is currently the gold standard for template quantification. The efficacy of this approach is limited by the necessary assumption that all samples are amplified with the same efficiency. To overcome this limitation, a new method has been proposed in this contribution for quantitative PCR with internal standard. Unlike existing methods based upon analysis of amplification profile position, the new method tries to determine the initial quantity of the target template in a sample from the fluorescence spectrum measured at a certain point during its PCR reaction. There is no unrealistic prerequisite (e.g., constant amplification efficiency) for the successful application of the new method. The performance of the new method was evaluated by the quantification of KRAS gene in HepG2 samples. Quantitative results with recovery rates in the range of 91.2–118% were achieved by the new method. It is reasonable to expect that the new method would have a place in real-time quantitative PCR, thanks to its features of no unrealistic prerequisite, sound theoretical basis, good performance, and implementation simplicity.

Therapeutic efficacy of artemether-lumefantrine in the treatment of uncomplicated Plasmodium falciparum malaria in Chewaka District, Ethiopia

BackgroundThe efficacy of artemether-lumefantrine (AL) for treatment of uncomplicated Plasmodium falciparum malaria in south-western Ethiopia is poorly documented. Regular monitoring of drug efficacy is an important tool for supporting national treatment policies and practice. This study investigated the therapeutic efficacy of AL for the treatment of P. falciparum malaria in Ethiopia.MethodsThe study was a one-arm, prospective, evaluation of the clinical and parasitological, responses to directly observed treatment with AL among participants 6 months and older with uncomplicated P. falciparum malaria. Real-time polymerase chain reaction (PCR) and nested PCR reaction methods were used to quantify and genotype P. falciparum. A modified protocol based on the World Health Organization 2009 recommendations for the surveillance of anti-malarial drug efficacy was used for the study with primary outcomes, clinical and parasitological cure rates at day-28. Secondary outcomes assessed included patterns of fever and parasite clearance. Cure rate on day-28 was assessed by intention to treat (ITT) and per protocol (PP) analysis. Parasite genotyping was also performed at baseline and at the time of recurrence of parasitaemia to differentiate between recrudescence and new infection.ResultsOf the 80 study participants enrolled, 75 completed the follow-up at day-28 with ACPR. For per protocol (PP) analysis, PCR-uncorrected and-corrected cure rate of AL among the study participants was 94.7% (95% CI 87.1–98.5) and 96% (95% CI 88.8–99.2), respectively. For intention to treat (ITT) analysis, the cure rate was 90% (95% CI 88.8–99.2). Based on Kaplan–Meier survival estimate, the cumulative incidence of failure rate of AL was 3.8% (95% CI 1.3–11.4). Only three participants 3.8% (95% CI 0.8–10.6) of the 80 enrolled participants were found to be positive on day-3. The day three-positive participants were followed up to day 28 and did not correspond to treatment failures observed during follow-up. Only 7.5% (6/80) of the participants were gametocyte-positive on enrollment and gametocytaemia was absent on day-2 following treatment with AL.ConclusionsThe therapeutic efficacy of AL is considerably high (above 90%). AL remained highly efficacious in the treatment of uncomplicated malaria in the study area resulted in rapid fever and parasite clearance as well as low gametocyte carriage rates despite the use of this combination for more than 15 years.

Prevalence of Adhesion Genes fnbA & fnbB of Staphylococcus aureus Isolated from Dental Patients Attending Dentistry Department of Tehsil Headquarter Hospital Pasrur, Pakistan

Introduction: The present study was intended to isolate, characterize and investigate the prevalence of virulence genes encoding fibronectin (fnbA and fnbB) adhesive molecules of S. aureus from dental patients attending outpatient department of THQ Hospital Pasur.
 Methodology: A total of 100 oral samples were collected from dental patients, pure cultures was segregated and identified through conventional microbiological methods to evaluate the prevalence of S. aureus. Isolates were further characterized by using specific primers for genotype fnbA and fnbB.
 Results: Results of the study declared that 68 samples were positive (68%) for staphylococcus aureus on the basis of growth on selective media and appearance of typical colonies supported by gram staining. These gram positive staphylococci were positive (86.7%) in coagulase and catalase testing. The results of polymerase chain reaction (PCR) revealed that 47 (69.1%) and 23 (33.8%) isolates showed amplification with type specific primer 23Sr RNA and NUC gene respectively. Furthermore, fnbA and fnbB type specific genes of S. aureus did not show any amplification in PCR reaction.
 Conclusion: Irrespectively the data in the present study showed the prevalence of S. aureus is significantly high in males and of age group of 20-40 years but no positive result was found for prevalence of fnbA and fnbB genes. All the S. aureus isolates were highly sensitive to vancomycin, linezolid and clindamycin.

Trypanosomes of vectors and domestic dogs in Trypanosoma cruzi transmission areas from Brazilian southwestern amazon: New mammalian host for Trypanosoma janseni

Trypanosoma cruzi is a widespread protozoan in Latin America causing Chagas disease in humans and able to infect several other mammal species. The objective of this study was to investigate the T. cruzi infection in triatomine fauna as well as in dogs from distinct areas of Acre, western Brazilian Amazonia, which recently reported acute cases of human CD as well as an area that have not notify this disease recently. Triatomines were collected and the intestinal contents were evaluated for the presence of trypanosomatids by optical microscopy and polymerase chain reaction (PCR) targeting the mini-exon gene. Blood smear, hemoculture, PCR and serology were performed in the studied mammals. Fecal content of four triatomines were positive (11.6%) in the fresh examination. Molecular analysis identified Trypanosoma cruzi TCI in two specimens. Blood samples from 90 dogs were obtained. Trypanosoma sp. was observed in six blood smears (6/83, 7.22%). Seropositivity for T. cruzi was 8/89 (8.98). One dog's hemoculture was obtained and characterized as T. rangeli. PCR reactions in blood clots resulted in one positive dog (1/75, 1.3%) infected by T. janseni, providing a new mammalian host for a recently described Trypanosoma species. The results demonstrate the low exposition and prevalence for T. cruzi suggesting that dogs are not important to T. cruzi transmission cycle in the studied áreas.

Application of 16S rRNA gene sequencing in Helicobacter pylori detection.

Helicobacter pylori is one of the major stomach microbiome components, promoting development of inflammation and gastric cancer in humans. H. pylori has a unique ability to transform into a coccoidal form which is difficult to detect by many diagnostic methods, such as urease activity detection, and even histopathological examination. Here we present a comparison of three methods for H. pylori identification: histological assessment (with eosin, hematoxylin, and Giemsa staining), polymerase chain reaction (PCR) detection of urease (ureA specific primers), and detection by 16S rRNA gene sequencing. The study employed biopsies from the antral part of the stomach (N = 40). All samples were assessed histologically which revealed H. pylori in eight patients. Bacterial DNA isolated from the bioptates was used as a template for PCR reaction and 16S rRNA gene sequencing that revealed H. pylori in 13 and in 20 patients, respectively. Thus, 16S rRNA gene sequencing was the most sensitive method for detection of H. pylori in stomach biopsy samples.

Portable and Battery-Powered PCR Device for DNA Amplification and Fluorescence Detection.

Polymerase chain reaction (PCR) is a technique for nucleic acid amplification, which has been widely used in molecular biology. Owing to the limitations such as large size, high power consumption, and complicated operation, PCR is only used in hospitals or research institutions. To meet the requirements of portable applications, we developed a fast, battery-powered, portable device for PCR amplification and end-point detection. The device consisted of a PCR thermal control system, PCR reaction chip, and fluorescence detection system. The PCR thermal control system was formed by a thermal control chip and external drive circuits. Thin-film heaters and resistance temperature detectors (RTDs) were fabricated on the thermal control chip and were regulated with external drive circuits. The average heating rate was 32 °C/s and the average cooling rate was 7.5 °C/s. The disposable reaction chips were fabricated using a silicon substrate, silicone rubber, and quartz plate. The fluorescence detection system consisted a complementary metal-oxide-semiconductor (CMOS) camera, an LED, and mirror units. The device was driven by a 24 V Li-ion battery. We amplified HPV16E6 genomic DNA using our device and achieved satisfactory results.

A Miniaturized Device for Performing PCR, Integrated with an Electrochemical DNA Biosensor for Detection of Corynebacterium Diphtheriae

Introduction Corynebacterium diphtheriae by secreting harmful toxin causes lethal disease called diphtheria. It occurs mainly in underdeveloped areas of the world with low vaccination rate [1]. Rapid detection of this pathogen is crucial to prevent serious health complications of the patient and spreading of the epidemic. As an alternative to time-consuming phenotypic analysis, a device has been designed, which allows the fast detection of the pathogen based on biological interactions at the molecular level, which reduces the risk of error. This device consists of a microfluidic system for performing a fast polymerase chain reaction (PCR), which is integrated with a DNA biosensor. It enables fast and unambiguous detection of bacteria in the sample, therefore it can be potentially used as a point-of-care device. Microfluidic PCR device The concept of rapid bacterial analysis is based on the amplification of a DNA fragment specific for a pathogen followed by its fast detection with e.g. biosensor. A miniaturized system for carrying out PCR was developed. The geometry of the microchannels was designed using CAD software and then micromilled in poly(methyl methacrylate). The microsystem was connected with miniaturized peristaltic pump and with a valve to enable automatic introduction, circulation and removal of the solution from the system. Moreover, a set of six heaters was used to divide the microsystem into three temperature zones. One cycle of the solution in a microchip corresponded with one reaction cycle. Temperatures of three zones were measured and controlled by resistance temperature sensor and set to the suitable values for following reaction steps: denaturation, annealing and elongation. Using the microsystem, any number of reaction cycles could be carried out and peristaltic pump enabled the medium flow in wide range (4 - 150µL/min). A number of both standard and asymmetric PCR reactions were carried out in the microchip using various polymerases, of which the Phire polymerase gave the most promising results. The products were analyzed by electrophoretic techniques and compared with the results of reactions carried out in thermocyclers. The ultimate goal is direct detection of the reaction product using a DNA biosensor. DNA biosensor An electrochemical DNA biosensor has been developed as detection part of a device. Its receptor layer was based on a hairpin like DNA probe labeled with methylene blue (MB). After the asymmetric PCR reaction the hybridization process took place which results in formation of double stranded DNA. The changes in hairpin like structure influenced the registered current and the same the biosensor response, proportional to PCR product concentration [2]. Results and Conclusions The miniaturized device consisting of a microchip with designed channel geometry, connected to the pump and valve was constructed. It enabled to automatically control the flow of a solution in a microchip and to carry out any number of reaction cycles. The reaction mixture changed its temperature rapidly when it flowed between different zones of the microchip, which contributed to a significant reduction in the reaction time. Asymmetric PCR was successfully carried out using the designed device in just 15 minutes. The electrochemical genosensor enabled detection of ssDNA, and its electrochemical signal was directly proportional to the concentration of the analyte in 0.0125-0.25µmol/L range. Studies on the effects of different factors, such as probe sequence, temperature, magnesium ion concentration and addition of dimethyl sulfoxide on electrochemical signal of a biosensor have also been performed.The constructed microchip allowed to significantly shorten the PCR reaction time, when compared with the thermocycler. Integration of microPCR with the genosensor might results in construction of fully automatized portable device enabling rapid and reliable pathogen detection. This type of instrument could be used in medical diagnostics as the point-of-care device contributing to the rapid detection of the disease, hence preventing the spread of the epidemic [3].

Transformation using RNAi technology for developing potato lines resistance to late blight (Phytophthora infestans)

Phytophthora infestans (Mont.) de Bary causes late blight, a major disease of potato and tomato that causes production loss at about 10–100% in Indonesia. Breeding for late blight resistance in potato has been carried out in this country to minimize fungicide application. The objective of this study was to generate potato lines resistant to P. infestans through RNAi method. A series of studies were conducted, including confirmation of RNAi construct insertion in Agrobacterium tumefaciens and PCR analysis, transformation of two potato cultivars using A. tumefaciens carrying the RNAi fragment, in addition to selection and molecular analysis of selected plantlet using Polymerase Chain Reaction (PCR). The RNAi plasmid construct was confirmed via PCR analysis using specific primers for 35s and Tnos fragment, and resulted in 500 bp and 250 bp for fragment of 35s and Tnos, respectively. Transformation was performed on 733 Granola’s internode explants and 569 Atlantic’s internode explants. The transformation process produced 282 explants from Atlantic, while Granola did not produce any transformants. The level of transformation efficiency of Atlantic on selection medium containing hygromycin was 49.61%. Following regeneration step, the 282 selected explants produced 167 plantlets. Based on PCR reaction using specific primers for hpt gene, 14 plantlets were PCR positive and contained hpt fragment. Overall, Agrobacterium tumefaciens-mediated transformation on potato internode explants was successful. Therefore, the selected transformants should be further tested using bioassay for resistance to P. infestans.

ZnO Nanolower-Based NanoPCR as an Efficient Diagnostic Tool for Quick Diagnosis of Canine Vector-Borne Pathogens.

Polymerase chain reaction (PCR) is a unique technique in molecular biology and biotechnology for amplifying target DNA strands, and is also considered as a gold standard for the diagnosis of many canine diseases as well as many other infectious diseases. However, PCR still faces many challenges and issues related to its sensitivity, specificity, efficiency, and turnaround time. To address these issues, we described the use of unique ZnO nanoflowers in PCR reaction and an efficient ZnO nanoflower-based PCR (nanoPCR) for the molecular diagnosis of canine vector-borne diseases (CVBDs). A total of 1 mM of an aqueous solution of ZnO nanoflowers incorporated in PCR showed a significant enhancement of the PCR assay with respect to its sensitivity and specificity for the diagnosis of two important CVBDs, Babesia canis vogeli and Hepatozoon canis. Interestingly, it drastically reduced the turnaround time of the PCR assay without compromising the yield of the amplified DNA, which can be of benefit for veterinary practitioners for the improved management of diseases. This can be attributed to the favorable adsorption of ZnO nanoflowers to the DNA and thermal conductivity of ZnO nanoflowers. The unique ZnO nanoflower-assisted nanoPCR greatly improved the yield, purity, and quality of the amplified products, but the mechanism behind these properties and the effects and changes due to the different concentrations of ZnO nanoflowers in the PCR system needs to be further studied.

Principle of Emulsion PCR and Its Applications in Biotechnology

Emulsion polymerase chain reaction (PCR) is performed on compartmentalized DNA, allowing a large number of PCR reactions to be carried out in parallel. Emulsion PCR has unique advantages in DNA amplification. It can be applied in many molecular biological assays, especially those requiring highly sensitive and specific DNA amplification. This review discusses the principle of emulsion PCR and its applications in biotechnology. Related technologies are also discussed.

Buğdayda PCR'a dayalı teknikler için DNA ekstraksiyon protokollerinin karşılaştırılması

Molecular biology and genetic studies requires high quality DNA isolated from plants. Therefore, isolation of pure and high quality DNA in a short time is important. However, since different plants have different chemical composition, in even close species need specific genomic DNA isolation protocol. There are a large number of protocols for wheat. Therefore, which method is more applicable, safer and easier should be determined. For this purpose three DNA extraction protocols were used. The DNA samples extracted from these protocols were compared for their purity and concentrations. The quality and amount of DNA obtained was determined by NanoDrop Spectrophotometer (A260/280 absorbance) and agarose gel electrophoresis. Additionally DNAs extracted were amplified by polymerase chain reactions (PCR) and the suitability of isolated DNAs for PCR reactions were also determined. The results show that with the CTAB DNA extraction methods were extracted the highest DNA concentrations in wheat ( Triticum avestivum L. ) compared to other protocols.

Rapid in vitro production of single-stranded DNA.

There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40–50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 μl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.

Improving detection of JC virus by ultrafiltration of cerebrospinal fluid before polymerase chain reaction for the diagnosis of progressive multifocal leukoencephalopathy

BackgroundProgressive multifocal leukoencephalopathy (PML) is a demyelinating disorder caused by JC virus (JCV). Although detecting JCV DNA in the cerebrospinal fluid (CSF) by real-time polymerase chain reaction (PCR) is useful, diagnosis is difficult when JCV concentrations are low. We therefore aimed to lower the detection limit of real-time PCR testing by enriching JCV in the CSF via ultrafiltration.MethodsVirus suspensions and CSF specimens from 20 untreated patients with suspected PML were collected and total DNAs were extracted. The JCV large T gene was detected by quantitative real-time PCR under condition with and without prior centrifugal ultrafiltration.ResultsThe JCV DNA was reliably detected to a lower limit of 10 copies/mL of virus suspension by real-time PCR with ultrafiltration. When using this method, the quantity of JCV DNA per PCR reaction increased 3.2- to 8.7-fold compared with the standard procedure. Seven patients were positive for JCV when using the standard procedure, and an additional patient was positive when using ultrafiltration. All JCV-positive patients had neurological features and magnetic resonance imaging findings compatible with PML.ConclusionsThe detection limit of JCV DNA by real-time PCR can be lowered by viral enrichment using ultrafiltration. Our simple protocol offers a valuable tool for PML diagnosis when extremely low copy numbers of JCV are released into the CSF or when brain biopsy is not feasible.

An Improved Multiplex Polymerase Chain Reaction (PCR) Assay for the Identification of Mosquito (Diptera: Culicidae) Blood Meals.

The analysis of vertebrate blood meals serves as an integral component of vector incrimination studies where feeding preferences and host associations influence vector-borne disease transmission. Diagnostic polymerase chain reaction (PCR)-based techniques have been widely used to determine host associations, yet applications for Culex (Diptera: Culicidae), which feed primarily on bird populations, have been limited by multistep PCR techniques that approach each potential host species singly. As a result, we have developed a multiplexed primer set targeting mitochondrial cytochrome b sequences that can distinguish human, bird, and mammalian host blood meals in a single PCR reaction, an improvement over previous analyses relying on single primers or other multiplex primer approaches through the inclusion of avian primers. To validate this new methodology, we demonstrate its application on blood samples as well as field-collected Culex samples. Although designed for applications with mosquito vectors, this multiplex PCR assay is not mosquito-specific, and should serve as a valuable tool for identifying the blood meals of other blood-feeding arthropods, contributing greatly to the study of vector-borne disease.

Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.

Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30–45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories.

DNA Barcode Quantification As a Robust Tool for Measuring Mixing Ratios in Two-Component Systems.

For many manufacturing processes, correct mixing compositions are crucial to guarantee product quality. However, the analysis of mixing ratios based on component balances can be challenging and requires extensive infrastructure. DNA barcodes have been previously proposed as low-cost markers for product authenticity, and we show here that the quantification of such barcodes via a quantitative real-time polymerase chain reaction (PCR) enables the determination of mixing ratios in a range of liquid and polymeric products. To enable the distribution of the DNA within the various matrixes, the biochemical is encapsulated in silica nanoparticles and distributed within the matrix of the raw material. If both raw materials of a two-component mixture contain such barcodes, the composition of the mixture can be determined from the relative concentration of the barcodes via multiplex PCR reactions, irrespective of the sampling volume and for a wide range of initial barcode concentrations (10 ppm to 10 ppb). As an application example, we use the barcodes to determine the mixing ratios of cross-linked and multicomponent polysilicon products.