Successful Polymerase Chain Reaction Research Articles

Poly(3,4-dihydroxyphenylalanine)-modified cellulose paper for the extraction of deoxyribonucleic acid by a laboratory-built automated extraction device

The success of polymerase chain reaction (PCR) depends on the quality of deoxyribonucleic acid (DNA) templates. This study developed a cost-effective and eco-friendly DNA extraction system utilizing poly(3,4-dihydroxyphenylalanine)-modified cellulose paper (polyDOPA@paper). PolyDOPA@paper was prepared by oxidatively self-polymerizing DOPA under weak alkaline conditions and utilizing the adhesive property of polyDOPA on different materials. Compared to the uncoated cellulose paper, polyDOPA coating significantly enhances DNA adsorption owing to its abundant amino, carboxyl, and hydroxyl moieties. The DNA extraction mechanism using polyDOPA@paper was discussed. The maximum adsorption capacity of polyDOPA@paper for DNA was 20.7 μg cm−2. Moreover, an automated extraction system was designed and fabricated using 3D printing technology. The device simplifies the operation and ensures the reproducibility and consistency of the results. More importantly, it eliminates the need for specialized training of operators. The feasibility of the polyDOPA@paper-based automated extraction system was evaluated by quantitatively detecting Escherichia coli in spiked milk samples via a real-time PCR. The detection limit was 102 cfu mL−1. The results suggest that the system would have significant potential in detecting pathogens.

A small review on polymerase chain reaction for the detection of Salmonella species

Salmonella identification from blood samples is crucial for rapid detection and efficient medication of typhoid and paratyphoid fever. Because of its remarkable sensitivity and specificity, polymerase chain reaction (PCR) is a broadly applied technology. The goal of this analysis of 16 papers concentrating on PCR-based Salmonella species identification in blood samples is to identify the most common and successful PCR techniques. The review covers a variety of PCR methods, such as one-step differential detection PCR, nested PCR, multiplex PCR, and real-time PCR. The effectiveness of many PCR primers, including those for the flagellin gene, hilA gene, invA gene, and iroB gene, in detecting Salmonella was examined. The examined studies consistently showed that the PCR techniques used had good sensitivity (95%–100%) and specificity (97%–100%). In addition, PCR was effectively used by the researchers to identify particular species of Salmonella serovars, which comprise Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis. Notably, multiplex PCR became a useful technique for detecting many Salmonella serovars at the same time. The use of PCR in identifying antibiotic resistance in Salmonella isolates is also emphasized in the review. The collective results highlight the remarkable specificity and sensitivity of PCR-based techniques for Salmonella species identification from blood samples. Of them, real-time PCR and multiplex PCR are the most widely used because of their increased efficiency, sensitivity, and specificity.

BRAF testing modalities in histiocytic disorders: Comparative analysis and proposed testing algorithm.

Understanding of histiocytic disorders has been revolutionized by demonstration of mitogen-activated protein kinase (MAPK) pathway mutations, most commonly BRAFV600E. The optimal testing strategy to assess BRAFV600E is unknown. We aimed to compare performance of testing modalities, to propose a framework for evaluation of BRAFV600E mutation status in histiocytic disorders. We retrospectively reviewed patients with histiocytic disorders and BRAF mutation testing on a lesional tissue specimen. In 120 patients, BRAF assessment included immunohistochemistry (IHC) in 97 (80.2%), polymerase chain reaction (PCR) in 35 (28.9%), and next-generation sequencing (NGS) in 62 (51.2%). Forty-five underwent both NGS and IHC. With NGS as the gold standard, the sensitivity and specificity of IHC were 82.4% and 96.4%. Three false negatives were observed in biopsy specimens with low BRAFV600E variant allele frequency or decalcified tissue. One false-positive IHC was observed in a lung biopsy specimen, likely due to antibody cross-reactivity with respiratory cilia. Among 14 with successful NGS and PCR, a single discordance was observed. Two PCR-to-IHC discrepancies were observed, including one other false-positive IHC. Immunohistochemistry was highly specific for detection of BRAFV600E. Main caveats were false negatives and lack of detection of non-BRAFV600E mutations. We propose the use of IHC as initial screening in general practice with reflex molecular testing if negative.

Comparison of different DNA preparatory methods for development of a universal direct PCR-RFLP workflow for identification of meat origin in food products

The quality and quantity of the extracted DNA are two key aspects for a successful PCR (Polymerase Chain Reaction) amplification. Moreover, reduction in time and cost required for DNA extraction are also two considerable factors, in cases when large number of samples are to be analyzed within a limited time-frame and budget. Accordingly, the aim of this study was to compare and optimize performance of five different DNA extraction methods by boiling meat tissues from cattle, buffalo, sheep, goat, chicken, camel, horse and dog in PBS (Phosphate Buffer Saline), distilled water, alkaline lysis buffers 1, 2 or 3. The results indicated that the boiling of meat and its products in alkaline lysis buffers was a good method to extract crude DNA. The optimized crude DNA extraction protocol was coupled with PCR-RFLP (Restriction Fragment Length Polymorphism) analysis for meat species differentiation. This developed workflow was tested on fifty-three commercial beef and mutton samples, out of which three samples were found to be adulterated. In conclusion, the rapid crude DNA extraction protocol has led to the development of a direct PCR-RFLP workflow that is simple, time-saving and cost-effective for PCR-based identification of different meat species.

In Vitro Isolation and Molecular Characterization of Genomic DNA from Ancient Human Long and Hip Bones

Background: A simple and effective modified ethanol precipitation-based protocol was described for the extraction of genomic DNA from ancient human bones. The qualitative and quantitative evaluation of genomic DNA was done based on DNA purity (260/280) and the PCR method. Method and Materials: In this study, a total of 50 embalmed ancient bones, including 20 long and 30 hip bone samples, were taken for genomic DNA extraction. The efficiency of the genomic DNA extraction was demonstrated on >50-year-old ancient human HIP and long bone samples. In vitro quantitative and qualitative analysis of extracted genomic DNA was performed by 0.8% agarose gel electrophoresis and PCR amplification. To assess the quality of extracted genomic DNA, a mitochondrial-specific ATPase6 gene primer was used to obtain sequence information of 675 bp. Result: Our data show that a concentration of genomic DNA between 1.6 and 2.0 at 260/280 resulted in successful PCR amplification. Our results demonstrated that the extraction of DNA from ancient bone samples with a manual approach will increase the amplification efficiency of the polymerase chain reaction (PCR). Conclusion: In the present study, a simple, time-saving, and cost-effective protocol is described for the extraction of genomic DNA from ancient human bones. Further, we believe the extraction of genomic DNA from ancient bone samples with this approach will increase the success rate of PCR amplification.

Myostatin (MSTN) Gene Detection in Nisi Chicken Using Real-Time PCR

The myostatin (MSTN) gene provides instructions for making myostatin, which helps control the growth and development of tissue throughout the body and limits muscle growth. Myostatin has been studied in mice, cows, and other animals. However, the MSTN gene has not been studied in the nisi chicken. Further, the lack of information using the real-time polymerase chain reaction (PCR) to detect the MTSN gene is still to be explored. Accordingly, this study aimed to detect the MSTN gene in nisi chickens, one of the local-endemic Indonesian chickens, using real-time PCR. DNA of twenty-five male chickens was extracted using an automatic DNA extraction tool, and the MSTN gene was analyzed by real-time PCR Rotor-Gene 5 Plex. Ct and Tm values were indicators of the successful amplification of the MSTN gene. The results showed that the MSTN gene was detected by Ct 16.00 and Tm 80.00, respectively. The study novelty is based on the nisi chicken as the local-endemic chicken from Indonesia as the study objective and the application of the real-time PCR as a method to detect the MSTN gene. From this study, we concluded that real-time PCR is a powerful method to detect the MSTN gene of the nisi chicken. MSTN gene detection using real-time PCR in nisi chickens is a step forward in molecular biology research to identify genetic diversity that can be one of the genetic markers in species DNA research. The aspect that is novel in this study lies in the use of the real-time PCR method in conducting tests to detect DNA species in nisi chickens. The lack of information in the field of research using this technique makes this research essential to be a source of information and reference in similar studies and one of the genetic markers that can apply to other studies in the field of species diversity. This study uses a qualitative testing method with real-time PCR. The interpretation of the results is based on the Ct and Tm values detected during amplification using real-time PCR, where the target gene detected is the MSTN gene. Ct and Tm values were used to see the success of real-time PCR in detecting target genes. Based on the results of real-time PCR, the MSTN gene was detected at Cy 16.00 and Tm. 80.00. In conclusion, the MSTN gene that was the target in this study was detected, which was characterized by amplification of the target DNA as indicated by the Ct and Tm values in real-time PCR.

Collagen-VI Specific Primer Design Identification in Rats (Rattus norvegicus) Pancreas

Type 2 diabetes mellitus, the most common diabetes type characterized by hyperglycemia, is caused by abnormal secretion and activity of pancreatic insulin enzymes. The extracellular matrix (ECM) plays a vital role in keeping β pancreatic cells intact and undissociated. The ECM in the pancreas can play a role in influencing insulin function and production. The most abundant ECM in the pancreas is collagen type VI. Collagen type VI has an essential role in the survival of pancreatic islet cells, including pancreatic β cells. Nowadays, Polymerase Chain Reaction (PCR) technology is widely utilized for molecular biology analysis. One of the most critical factors for successful PCR is designing the correct specific PCR primers. The objective of this study was to design a specific primer for collagen VI in the pancreas of Rattus norvegicus. The primer was designed and analyzed using MEGA.11, primer three-plus, and primer-BLAST. Five primer pairs were analyzed based on the characteristics of primer length, product amplicon length, Tm value, GC percentage, and secondary structure. Primer pair 3 (F:5’-TGTTTGGCTTTGTCGCGGGC-3’ and R:5’-TTGTTGCTGCCGACACTGGC-3’); Col6a2 (F:5’-TGTGGTCAACAGGCTGGGCG-3’ and R:5’-TCTGGCGCCGGCTCTCTTTG-3’) were considered as the best primer for the Collagen VI expression detection from the pancreas of Rattus norvegicus, which produce amplicon about 250pb and 245pb, respectively.

A survey of the effects of common illicit drugs on forensic DNA analysis

Profiling of DNA associated with illicit drug packages and paraphernalia is a common investigative tool. In addition, research is being conducted regarding the analysis of trace DNA present within illicit drugs and on capsules. The application of trace DNA analysis to illicit drugs has the potential to identify individuals involved in their manufacture and distribution. However, the inhibitory effects of illicit drugs and related compounds on downstream DNA analysis has not yet been investigated. If drug-induced polymerase chain reaction (PCR) inhibition occurs, the quality or informativeness of the resultant DNA profile may be impacted. In this study, the effects of a range of drugs, diluents, adulterants, and synthetic precursors on both quantitative PCR (qPCR) data and short tandem repeat (STR) DNA profiling results were examined. Twenty-two compounds representative of drug compounds and adulterants which may be encountered in drug seizures were spiked with 1 ng/μL and 0.05 ng/μL of control DNA and underwent DNA quantification using Quantifiler™ Trio. A subset of 13 compounds, including the majority that indicated potential inhibition in Quantifiler™ Trio, underwent STR profiling with VeriFiler™ Plus to determine if inhibition also occurred at this stage. The effect of diluting the DNA extract on the extent of inhibition of STR profiling was also investigated. Internal PCR controls within the qPCR were not a reliable indicator of inhibition, although suppression of the short and long autosomal fragments was observed in the presence of many compounds, and four compounds gave inconclusive results. STR internal quality controls indicated inhibition in 5 of the 13 compounds, however, profiles were affected by the presence of 11 of the 13 compounds in various ways such as a decreased average relative fluorescence units (RFU), drop out of certain alleles (some based on allele size range of locus) leading to a decreased likelihood ratio (LR), an increase in the proportion of stutter peaks and the presence of split or shoulder peaks. All profiles improved following a dilution of the compound in the PCR and allowing the generation of LR values in excess of 1 × 1025, indicating inhibition occurred rather than DNA degradation. The data obtained show that removal of some of these compounds is required through an effective DNA extraction process for successful downstream trace DNA profiling. Upon successful PCR, the resultant DNA profiles provide the opportunity for opening new investigative avenues for law enforcement agencies.

A plasmonic gold nanofilm-based microfluidic chip for rapid and inexpensive droplet-based photonic PCR

Polymerase chain reaction (PCR) is a powerful tool for nucleic acid amplification and quantification. However, long thermocycling time is a major limitation of the commercial PCR devices in the point-of-care (POC). Herein, we have developed a rapid droplet-based photonic PCR (dpPCR) system, including a gold (Au) nanofilm-based microfluidic chip and a plasmonic photothermal cycler. The chip is fabricated by adding mineral oil to uncured polydimethylsiloxane (PDMS) to suppress droplet evaporation in PDMS microfluidic chips during PCR thermocycling. A PDMS to gold bonding technique using a double-sided adhesive tape is applied to enhance the bonding strength between the oil-added PDMS and the gold nanofilm. Moreover, the gold nanofilm excited by two light-emitting diodes (LEDs) from the top and bottom sides of the chip provides fast heating of the PCR sample to 230 °C within 100 s. Such a design enables 30 thermal cycles from 60 to 95 °C within 13 min with the average heating and cooling rates of 7.37 ± 0.27 °C/s and 1.91 ± 0.03 °C/s, respectively. The experimental results demonstrate successful PCR amplification of the alcohol oxidase (AOX) gene using the rapid plasmonic photothermal cycler and exhibit the great performance of the microfluidic chip for droplet-based PCR.

Comparison of methods to extract PCR-amplifiable DNA from fruit, herbal and black teas

The success of polymerase chain reaction (PCR) assay depends on template deoxyribonucleic acid (DNA) being sufficient with respect to both quantity and quality. Some biological materials contain compounds which inhibit the functioning of DNA polymerase and thus need to be removed as part of the DNA extraction procedure. The aim of the present experiments was to optimise the process of DNA isolation from various types of black, fruit and herbal teas. A comparison was made between two cetyltrimethylammonium bromide (CTAB)-based protocols and two commercially available DNA purification kits. The yield and integrity of the extracted DNA were monitored both spectrophotometrically and using agarose gel electrophoresis. The presence/absence of inhibitors in the DNA preparations was checked by running quantitative real-time PCRs. The optimal protocol was deemed to be the CTAB method described in ISO 21571:2005, so this method is recommended for the routine sample analysis of tea products.

Development of a Rapid and Low-Cost Method for the Extraction of Dermatophyte DNA.

Background:Polymerase chain reaction (PCR) is the most optimized method for the rapid detection and analysis of any environmental or clinically significant organism. While PCR amplification directly from samples has been shown effective for several bacteria and viruses, for filamentous fungus and yeast, extraction of genomic DNA is a must. The extraction of DNA from fungal cultures is often reported using user-friendly commercially available kits, which are designed to decrease the time, extensive manual work in extraction procedures but are often expensive. Dermatophytes pose an added drawback to efficient DNA extraction due to their poor recovery on culture media and slow growth rate.Aims and Objectives:In the present study, we developed and validated a method for effective genomic DNA extraction from dermatophytes.Materials and Methods:DNA yield from standard dermatophytes extracted from spore suspensions and mycelia mat by commercially available kits was compared. A modified method using lyticase buffer and phenol-chloroform extraction was developed. The yield obtained was compared with the existing methods (kit-based method and cetyl trimethyl ammonium bromide method). The yield and quality of the total genomic DNA were estimated spectrophotometrically and by successful PCR amplification of the ITS region. The results were validated using 21 clinical isolates from recalcitrant dermatophytosis.Results:Minimal fungal DNA was obtained from the spores compared to that obtained from mycelial mat. Commercially available kits yielded lower amounts of DNA compared to the CATB method. The modified method developed in this study yielded better quality and quantity of DNA.Conclusion:Of the three extraction methods evaluated, the developed method gave significantly higher total genomic DNA yield and better purity than the reference methods. In addition, the turnaround time for DNA extraction was reduced to half based on modifications in culture conditions.

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.

Numerical method for optimization of thermal cycling in 3D-printed polymerase chain reaction device

A continuous-flow polymerase chain reaction (PCR) device with integrated heaters for DNA amplification was studied. We developed a numerical model to predict the temperature variations of a 3D-printed PCR device for different heater temperatures. The target temperatures at thermal zones for a successful PCR are 95 °C, 55 °C, and 72 °C for denaturation, annealing and extension, respectively. A numerical simulation was conducted to determine the heater temperatures required to compensate for heat loss. Our numerical model and optimization method were confirmed by comparison with the experimental results. Consequently, the compensated heater temperatures required to achieve the target temperatures are 97.9 °C, 56.6 °C and 74.1 °C. The proposed optimization method and numerical simulations will be useful in achieving highly effective thermal cycling in the 3D-printed PCR device.

Detection of cryIII gene in Local Isolate of Bacillus thuringiensis Using Polymerase Chain Reaction (PCR)

Bacillus thuringiensis is a biological biopesticide that was used for defense against pests. In B. thuringiensis there is a Cry protein that is only toxic to certain insects. This Cry protein is encoded by cry gene. There are many types of cry genes that have been identified, one of which is cryIII gene that is toxic to insects from the Coleoptera group as pests in sweet potato (Ipomoea batatas). The aim of this study was to amplify the cryIII gene from local isolate of B. thuringiensis. The method that can be used for cryIII gene amplification is Polymerase Chain Reaction (PCR). The primer pair is one of the important factors that determine the success of PCR. From a number of designed primers, the primer pair selected to be used in this study is Cry3B forward 5’-AAAGTGCGGCTATTCGACCA-3’ and Cry3B reverse 5’-CACTTCATCCTGTGACGCCT-3’. This primer pair successfully amplified cryIII gene and showed a DNA band with molecular size approximately 914 base pairs. Gradient PCR needs to be done for optimizing specific amplification of cryIII gene.
 
 Keywords: PCR, primer, sweet potato

MOLECULAR DETECTION AND CONTROLLING OF SEED-BORNE Colletotrichum spp. IN COMMON BEAN AND SOYBEAN

Seed-borne fungi cause several diseases of common bean (Phaseolus vulgaris L.) and soybean (Glycine max L.). The survey of seed-borne fungi of common bean and soybean was carried out on three Egyptian Governorates (Behera, Dakahlia, and Ismailia) in Egypt. Nineteen fungal species comprising therteen genera were isolated from the collected common bean and soybean seed samples, using standard blotter i,e. Alternaria alternata, Alternaria spp., Aspergillus niger, Aspergillus ochraceous, Aspergillus flavus, Botryodiplodia sp., Cladosporium sp., Colletotrichum lindemuthianum, Colletotrichum dematium, Fusarium solani, Fusarium moniliforme, Fusarum oxysporum, Macrophomina phaseolina, Myrothecium sp., Penicillium spp, Rhizoctonia solani, Stemphylium spp., Trichoderma spp. and Trichothecium sp. Pathogenicity tests proved that C. lindemuthianum and C. dematium were pathogenic to common bean and soybean. Polymerase chain reaction (PCR) has many beneficial characteristics that make it highly applicable for detecting Colletotrichum spp. of seeds. PCR diagnosis method and DNA extraction considered one of the most important steps and purity of DNA template for successful PCR assay. For the PCR amplification of C. lindemuthianum and C. dematium, two primers ClF4 and ClF5, stander blotter, agar plate, and deep freezing method were used as seed healthy testing methods. Stander blotter was proved the past and quickly method to detect seed-born of C. lindemuthianum and C. dematium. Trichoderma harzianum reduced linear growth for C. lindemuthianum and C. dematium followed by Trichoderma viride and Bacillus subtilis recording (3.3 cm and 3.9 cm, respectively) followed by Pseudomonas floursence that display a high linear growth (7.4 cm). On the other hand, fungicide Aetro 30% (Iprodione + Tebuconazole) recorded the least linear growth (0.9 cm) for C. lindemuthianum and C. dematium compared with negative control recorded (9.0 cm in diameter). Thymus plant extract caused the lowest liner growth (3.7 cm). Plant extract concentrations caused a significant reduction in the growth of C. lindemuthianum and C. dematium. The highest concentration (1.5 %) gave less fungal growth while the lowest one (0.5%) showed the highest fungal growth compared with negative control.

Comparison of DNA Isolation and Dominant and Co-dominant Molecular Markers to Reveal the Genetic Sex of Gallus domesticus (Domestic Chickens)

Purpose: The quality of DNA and reliability of molecular markers are crucial for the success of Polymerase Chain Reaction (PCR) based genetic sex determination. This study was aimed at investigating the optimum conditions for isolation of DNA from chicken blood and the reproducibility of dominant and co-dominant sex markers to be validated as a tool for successful sexing in avian research.Research Method: Efficacy of six different extraction procedures including manual and solution based commercial purification kit were evaluated with different combinations of initial blood, lysis buffer and protein denaturant in relation to the DNA yield and purity. Three primer sets namely CHD1, HUR 0423 and HUR 0424 were evaluated by PCR. Findings: The study results showed that 10µl of initial blood volume yields a significantly high DNA yield with high purity. Dominant marker HUR0424 showed to be a reliable marker system for the genetic sexing of domestic chickens over co-dominant markers.Research Limitation: For the accuracy of the results, protocols had to be followed at the same time and using same sample to avoid any errors.Originality/ Value: PCR based sexing is considered, the most accurate and inexpensive method and hence validation of the method is important for success of future avian research.

Extracting the invisible: obtaining high quality DNA is a challenging task in small arthropods.

BackgroundThe application of an appropriate extraction method is a relevant factor for the success of all molecular studies.MethodsSeven different DNA extraction methods suitable for high-throughput DNA sequencing with very small arthropods were compared by applying nine different protocols: three silica gel based spin methods, two cetyltrimethyl ammonium bromide (CTAB) based ones (one with an additional silica membrane), a protein precipitation method and a method based on a chelating resin (applying different protocols). The quantity (concentration) and quality (degradation, contamination, polymerase chain reaction (PCR) and sequencing success) of the extracted DNA as well as the costs, preparation times, user friendliness, and required supplies were compared across these methods. To assess the DNA quantity, two different DNA concentration measurements were applied. Additionally, the effect of varying amounts of starting material (different body sizes), variable lysis temperatures and mixing during DNA extraction was evaluated.ResultsAlthough low DNA concentrations were measured for all methods, the results showed that—with the exception of two methods—the PCR success was 100%. However, other parameters show vast differences. The time taken to perform DNA extraction varied from 20 min to 2.5 h (Chelex vs. CTAB) and the costs from 0.02 to 3.46 € (Chelex vs. QIAamp kit) per sample. High quality genomic DNA was only gained from four methods. Results of DNA quantity measurements further indicated that some devices cannot deal with small amounts of DNA and show variant results.DiscussionIn conclusion, using Chelex (chelating resin) turned out as a rapid, low-cost method which can provide high quality DNA for different kinds of molecular investigations.

Liquid marbles as biochemical reactors for the polymerase chain reaction.

The polymerase chain reaction (PCR) is a popular and well-established DNA amplification technique. Technological and engineering advancements in the field of microfluidics have fuelled the progress of polymerase chain reaction (PCR) technology in the last three decades. Advances in microfluidics-based PCR technology have significantly reduced the sample volume and thermal cycling time. Further advances led to novel and accurate techniques such as the digital PCR. However, contamination of PCR samples, lack of reusability of the microfluidic PCR platforms, complexity in instrumentation and operation remain as some of the significant drawbacks of conventional microfluidic PCR platforms. Liquid marbles, the recently emerging microfluidic platform, could potentially resolve these drawbacks. This paper reports the first liquid marble based polymerase chain reaction. We demonstrated an experimental setup for the liquid-marble based PCR with a humidity-controlled chamber and an embedded thermal cycler. A concentrated salt solution was used to control the humidity of the PCR chamber which in turn reduces the evaporation rate of the liquid marble. The successful PCR of microbial source tracking markers for faecal contamination was achieved with the system, indicating potential application in water quality monitoring.

Rapid species identification of sloth bears from non-invasive samples: a PCR-based assay

Once common throughout the Indian subcontinent, the sloth bear (Melursus ursinus) is restricted to certain areas and is classified as vulnerable by the International Union for Conservation of Nature. Habitat loss and fragmentation is the primary reason for population decline, but there is a dearth of information about the effects of fragmentation on connectivity and genetic variation. Non-invasive DNA samples are commonly used to investigate connectivity and monitor populations in the wild. We developed a polymerase chain reaction (PCR) –based technique to identify sloth bears from non-invasively collected samples. Three sets of primers were designed to amplify short fragments of mitochondrial DNA to reduce false negatives, which is an issue with non-invasively collected DNA. The PCR success rate in both fresh and dry samples was high (88%). Failure to amplify DNA from other co-occurring mammals confirmed species specificity. This approach provides an efficient method to identify sloth bear samples using non-invasively collected DNA.

Intrapartum group B Streptococcus screening in the labor ward by Xpert® GBS real-time PCR.

Group B Streptococcus (GBS) is the leading cause of neonatal infections in industrialized countries. Intrapartum antibiotic prophylaxis (IAP) given to colonized parturients is a key step for the prevention of neonatal early-onset infection. We compared the performances of Xpert® GBS polymerase chain reaction (PCR) (Cepheid, Sunnyvale, CA, USA) as a point-of-care system in labor wards to standard culture for intrapartum GBS detection. Pregnant women with a GBS-positive antenatal screening were prospectively included. A vaginal double swab was collected at the time of delivery for point-of-care Xpert® GBS PCR and GBS culture. A total of 565 pregnant women were included. Valid Xpert® GBS results were obtained for 488 (86.4%) women on the first attempt. Repeat testing improved the PCR success to 516 (91.3%) women. Among the 305 women positive for GBS by culture at delivery, only 238 (78.0%) were positive by Xpert® GBS PCR, cycle thresholds being correlated to culture quantification. Among 260 women negative for GBS culture, 56 (21.5%) were positive by Xpert® GBS PCR, including 50 where IAP was initiated before vaginal sampling. Overall, among the 565 women with GBS antenatal positive culture, only 335 (59.3%) were still positive at delivery whatever the technique used, resulting in unnecessary IAP for 40% of them. This large cohort study comparing intrapartum to antepartum GBS detection provides evidence that (i) Xpert® GBS PCR might be a valuable solution for intrapartum GBS detection compared to culture-based strategies and (ii) laboratory training of non-specialized staff is mandatory to reach the performances required for point-of-care tests.