Polymerase Chain Reaction Platform Research Articles

Latent Class Analysis for the Diagnosis of Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) is an opportunistic disease that lacks a gold-standard test. Nucleic acid amplification tests such as real-time polymerase chain reaction (PCR) demonstrate an excellent limit of detection (LOD), whereas antigenic methods are able to detect protein toxin. Latent class analysis (LCA) provides an unbiased statistical approach to resolving true disease. A cross-sectional study was conducted in patients with suspected CDI (N = 96). Four commercial real-time PCR tests, toxin antigen detection by enzyme immunoassay (EIA), toxigenic culture, and fecal calprotectin were performed. CDI clinical diagnosis was determined by consensus majority of 3 experts. LCA was performed using laboratory and clinical variables independent of any gold standard. Six LCA models were generated to determine CDI probability using 4 variables including toxin EIA, toxigenic culture, clinical diagnosis, and fecal calprotectin levels. Three defined zones as a function of real-time PCR cycle threshold (Ct) were identified using LCA: CDI likely (>90% probability), CDI equivocal (<90% and >10%), CDI unlikely (<10%). A single model comprising toxigenic culture, clinical diagnosis, and toxin EIA showed the best fitness. The following Ct cutoffs for 4 commercial test platforms were obtained using this model to delineate 3 CDI probability zones: GeneXpert®: 24.00, 33.61; Simplexa®: 28.97, 36.85; Elite MGB®: 30.18, 37.43; and BD Max™: 27.60, 34.26. The clinical implication of applying LCA to CDI is to report Ct values assigned to probability zones based on the commercial real-time PCR platform. A broad range of equivocation suggests clinical judgment is essential to the confirmation of CDI.

Wastewater-Based Epidemiology to monitor COVID-19 outbreak: Present and future diagnostic methods to be in your radar

The WHO has declared the COVID-19 epidemic on January 31, 2020. This virus has infected millions of people worldwide in just a few months. Shortly afterwards, the National Medical Products Administration (NMPA) announced nucleic acid testing as the gold standard for virus detection. Antibody testing is used as well as a supplementary test for suspected cases where nucleic acid detection was negative. In short, nucleic acid–based polymerase chain reaction (PCR) is the mainstream detection method for clinical samples as well as for the detection of SARS-CoV-2 in wastewaters. First data collected around the globe were reported in the last few months being part of the so-called Wastewater-Based Epidemiology (WBE) approach. Selection of concentration methods and primers, laboratory inter-comparison and various modalities of PCR detection of the virus in complex wastewater matrices were flagged up as main bullets that require urgent improvement. Novel approaches to enhance sensitivity, speed and automate streamlined virus detection will be discussed here as well. This list comprises devices mainly used for clinical purposes like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), Digital PCR, Lab-on-a-chip (LOC) and related platforms as well as Biosensors. The last part will be devoted to the identification of biomolecules to target Covid-19 outbreak based on inflammatory response biomarkers among others. To this end this opinion paper brings for discussion the issue of PCR detection and its limitations as well as new diagnostic methods in WBE.

Fast and reliable detection of carbapenemase genes in various Gram negatives using a new commercially available fluorescence-based real-time polymerase chain reaction platform

The Revogene® Carba C assay is a real-time polymerase chain reaction–based assay that runs on the microfluidic Revogene platform. It was recently designed for the detection of genes encoding the 5 major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48) from various Gram-negatives. A total of 145 clinical Gram-negative strains (96 carbapenemase producers and 49 non–carbapenemase producers) were tested. The overall sensitivity and specificity were 100%. All strains co-producing double carbapenemases have been correctly detected. All non–carbapenemase producers and nontargeted carbapenemase producers gave a negative result. The sample preparation was easy to handle, taking around 5 to 10 min per isolate, with a run time of approximately 70 min. This assay is a rapid, easy-to-perform, reliable tool to detect the most common carbapenemases, with excellent sensitivity and specificity regardless of the host bacteria. Given its user friendliness, simplicity, and short time to result, the Revogene® Carba C assay is suitable for microbiology laboratories.

Semen infections in men with primary infertility in the real-life setting

To study the impact of asymptomatic semen infections on seminal parameters in men presenting for primary couple's infertility. Cross-sectional study. Academic center. Socio-demographic, clinical, and laboratory data from 1689 infertile men were analyzed. Semen analysis was based on 2010 World Health Organization reference criteria. Each patient underwent semen culture test to identify common urogenital pathogens. Infections by Mycoplasma, Ureaplasma, and Chlamydia spp. were evaluated through a real time polymerase chain reaction platform. Descriptive statistics and linear and logistic regression models were used to test the association between semen infections and clinical, seminal, and hormonal parameters. Prevalence of asymptomatic semen infection and impact of semen infection on sperm parameters. Of 1689 men, 354 (21.0%) had an asymptomatic positive semen culture. Ureaplasma urealyticum (37.6%) was the most frequent single pathogen, followed by Enterobacteriaceae (any type; 24.8%), other pathogens (20.3%), Chlamydia trachomatis (3.4%) and Mycoplasma spp (3.4%). Positive semen cultures were associated with lower sperm concentrations (P<0.001) and progressive motility (P<.001). These latter findings were mostly particular to men with infections caused by Ureaplasma urealyticum compared with negative semen cultures. A positive semen culture was both univariably (P<.001) and multivariably (P=.04) associated with a lower sperm concentration. One out of five men presenting for a couple's primary infertility had asymptomatic semen infections, which were significantly associated with impaired sperm concentration. These observations point out the importance of an accurate investigation of semen infection in the everyday clinical practice diagnostic workup of infertile men.

A novel urinary mRNA signature using the droplet digital polymerase chain reaction platform improves discrimination between prostate cancer and benign prostatic hyperplasia within the prostate-specific antigen gray zone.

PurposeThe aim of this study was to identify a noninvasive urinary marker for prostate cancer (PCa) diagnosis and to validate the clinical performance of this novel urinary mRNA signature using the droplet digital polymerase chain reaction (ddPCR) approach.Materials and MethodsA gene expression microarray (HT-12, Illumina Inc., USA) was used to identify genes differentially expressed between 16 PCa and 8 benign prostatic hyperplasia (BPH) tissues; ddPCR (QX200; Bio-Rad Laboratories, USA) was carried out to quantify the expression of selected genes in urine. The urinary molecular PCa risk score (UMPCaRS) was calculated by using the sum of three upregulated genes as the numerator and the sum of three downregulated genes as the denominator. The diagnostic utility of the UMPCaRS was validated by using a screening set (10 PCa and 10 BPH samples) and a validation set (131 PCa and 105 BPH samples).ResultsThree upregulated genes (PDLIM5, GDF-15, THBS4) and three downregulated genes (UPK1A, SSTR3, NPFFR2) were selected from the microarray and subjected to ddPCR. The UMPCaRS for PCa in the screening and validation sets was significantly higher than that for BPH. For the validation set, the diagnostic accuracy of the UMPCaRS was comparable with that of prostate-specific antigen (PSA). Importantly, in the “PSA gray zone” (3–10 ng/mL), the AUC for the UMPCaRS was 0.843 and that for PSA was 0.628 (p<0.001).ConclusionsThe data demonstrate that the UMPCaRS is useful for discriminating between PCa and BPH in the “PSA gray zone”.

Rapid identification of Babesia canis and Babesia gibsoni (Asian genotype) in canine blood samples using a customized portable real-time PCR analyzer and TaqMan-based assay

Canine babesiosis is a serious infectious disease in subtropical and tropical regions. Typically, clinical detection of canine babesiosis is performed by blood smear observation or the traditional polymerase chain reaction (PCR). Herein, we developed a new TaqMan-based real-time PCR assay combined with a customized portable real-time PCR platform for a rapid and accurate detection of canine babesiosis. Two new primer/probe pairs (B18S and BITS1) were designed based on 18S ribosomal RNA and an internal transcribed spacer 1 (ITS1) sequence to differentiate Babesia canis and B. gibsoni (Asian genotype) DNAs from canine blood samples. Additionally, a corresponding customized compact real-time PCR platform with low 6-carboxyfluorescein fluorescence detection (≤5 nM), including a fast and accurate thermal cycling ability with a user-friendly interface for thermal control and data analysis, was designed for the limited space use. Both assays (B18S and BITS1) demonstrated a sensitivity of 100 copies/reaction based on the 95 % confidence interval evaluation method. The self-developed customized portable real-time PCR analyzer presented high repeatability and reproducibility with the TaqMan-based assay. Moreover, 501 clinical specimens were collected for evaluating the performance of the proposed PCR. The positive and negative predictive values were 90 % (18 of 20) and 100 % (226 of 226), respectively, for samples suspected with B. canis infection and 98 % (55 of 56) and 100 % (199 of 199), respectively, for samples suspected with B. gibsoni infection.

Microfluidic Array Chip for Parallel Detection of Waterborne Bacteria.

The polymerase chain reaction (PCR) is a robust technique used to make multiple copies of a segment of DNA. However, the available PCR platforms require elaborate and time-consuming operations or costly instruments, hindering their application. Herein, we introduce a sandwiched glass–polydimethylsiloxane (PDMS)–glass microchip containing an array of reactors for the real-time PCR-based detection of multiple waterborne bacteria. The PCR solution was loaded into the array of reactors in a single step utilising capillary filling, eliminating the need for pumps, valves, and liquid handling instruments. Issues of generating and trapping bubbles during the loading chip step were addressed by creating smooth internal reactor surfaces. Triton X-100 was used to enhance PCR compatibility in the chip by minimising the nonspecific adsorption of enzymes. A custom-made real-time PCR instrument was also fabricated to provide thermal cycling to the array chip. The microfluidic device was successfully demonstrated for microbial faecal source tracking (MST) in water.

Comparative spatial lipidomics analysis reveals cellular lipid remodelling in different developmental zones of barley roots in response to salinity.

Salinity‐induced metabolic, ionic, and transcript modifications in plants have routinely been studied using whole plant tissues, which do not provide information on spatial tissue responses. The aim of this study was to assess the changes in the lipid profiles in a spatial manner and to quantify the changes in the elemental composition in roots of seedlings of four barley cultivars before and after a short‐term salt stress. We used a combination of liquid chromatography–tandem mass spectrometry, inductively coupled plasma mass spectrometry, matrix‐assisted laser desorption/ionization mass spectrometry imaging, and reverse transcription – quantitative real time polymerase chain reaction platforms to examine the molecular signatures of lipids, ions, and transcripts in three anatomically different seminal root tissues before and after salt stress. We found significant changes to the levels of major lipid classes including a decrease in the levels of lysoglycerophospholipids, ceramides, and hexosylceramides and an increase in the levels of glycerophospholipids, hydroxylated ceramides, and hexosylceramides. Our results revealed that modifications to lipid and transcript profiles in plant roots in response to a short‐term salt stress may involve recycling of major lipid species, such as phosphatidylcholine, via resynthesis from glycerophosphocholine.

Simultaneous Detection of Exosomal Membrane Protein and RNA by Highly Sensitive Aptamer Assisted Multiplex–PCR

Exosomes, which are 30-150 nm extracellular vesicles containing a variety of biomolecules (i.e., proteins, RNA, DNA, and lipids), have emerged as important analytes in liquid biopsy. Although exosome detection offers a valuable chance to understand disease status in a multidimensional manner, comprehensive analysis of different exosomal biomolecules with limited specimens remains a challenge. Herein, we introduced an aptamer-based PCR (polymerase chain reaction) platform and realized highly sensitive detection of CD63 protein positive exosomes as low as 50 particles/μL. More importantly, simultaneous analysis of exosomal RNA and surface protein is available using this method. In a proof of concept experiment, simultaneous detection of two immunotherapy-related biomarkers, programmed death-ligand 1 (PD-L1) protein and indoleamine 2,3-dioxygenase 1 (IDO1) mRNA, was demonstrated with clinical samples. Our method provides a strategy for the comprehensive analysis of exosomes, with the advantages of high sensitivity and practicability.

Identification of Enteropathogens by Multiplex PCR among Rural and Urban Guatemalan Children with Acute Diarrhea.

Multiplex polymerase chain reaction (PCR) platforms have enhanced understanding of intestinal pathogens in low- and middle-income countries (LMICs). However, few such studies have been performed in Latin America, where poverty, poor sanitation, and undernutrition persist. Multiplex PCR (BioFire, Salt Lake City, UT) was used to identify viral, bacterial, and parasitic pathogens in stool collected on day 1 and 31 from children aged 6 to 35 months with acute, non-bloody diarrhea in two locations (rural and urban) in Guatemala. We analyzed correlation between pathogens and clinical, demographic, and socioeconomic variables; described patterns of pathogen acquisition, persistence, and clearance over the 30-day period; and calculated population attributable fractions (PAFs) for diarrheal causation for individual pathogens. We analyzed 316 subjects (144 urban; 172 rural) enrolled between March 2015 and January 2016. Rural subjects had significantly more malnutrition, animal exposure, and unimproved water/sanitation infrastructure. The majority of subjects had multiple pathogens/sample (4.8 rural and 2.7 urban). Few meaningful correlates were identified between individual pathogens and clinical, demographic, or environmental variables. Escherichia coli pathotypes, Shigella, Campylobacter, and Giardia had high rates of persistence between initial and 30-day follow-up. Statistically significant adjusted PAFs were identified for Campylobacter (14.9%, 95% CI: 3.2-23.1), norovirus (10.2%, 95% CI: 0.4-17.1), sapovirus (7.6%, 95% CI: 2.3-10.9), and adenovirus 40/41 (5.6%, 95% CI: 0.3-8.7). These observations further characterize the diversity and complexity of enteric pathogens in children in LMICs. Patterns of chronic symptomatic and asymptomatic infection among Latin American children are similar to those observed in other LMIC regions. Findings have direct implications for practitioners treating individuals with acute infectious diarrhea and should inform regional public health strategies.

Development of fast and sensitive protocols for the detection of viral pathogens using a small portable convection PCR platform.

One of the most crucial steps for preventing viral pandemics is the early detection of the causative virus on site. Various molecular and immunological approaches have been developed for virus detection. In this study, we investigated the utility of the recently introduced convection polymerase chain reaction (cPCR) platform for the rapid and sensitive detection of various animal viruses in the field, including the foot-and-mouth disease virus (FMDV) and avian influenza viruses (AIVs). Primer sets were designed to simultaneously detect two highly conserved regions of the FMDV, including the 5' untranslated region (5'-UTR) and 3D gene, and to specifically amplify the NP and hemagglutinin (HA) genes of H5 and H9 subtypes of AIVs. The portable cPCR system was able to amplify from as low as 1 to 10 copies of viral cDNAs in the singleplex mode and 10 to 100 copies of viral cDNAs in the duplex mode within 21min. Thus, our data suggest that the cPCR protocols developed in this study are highly sensitive and enable quick detection of animal viruses in biological samples.

Abstract 2455: Single-color accurate and precise quantification of wild-type versus mutant allele using a novel integrated digital PCR platform

Abstract In this study we present a single-step, one-color quantification of mutant versus wild-type allele using a novel integrated, clinic-optimized digital PCR (dPCR) platform. Allelic variants in DNA or RNA can be distinguished and quantified using Polymerase Chain Reaction (PCR) in concert with intelligent primer and probe design. Most commonly, a researcher or clinician will use unlabeled forward and reverse primers that bind to conserved regions outside of the allelic variation in addition to two differently labeled hydrolysis probes that bind at the region of allelic variation. Here we describe a novel method that leverages the precision of digital PCR (dPCR) technology and requires only one-color fluorescence utilizing probe-free, intercalating dye chemistry. The expense in developing or purchasing a complete PCR assay is normally embedded in the cost of the fluorescently labeled probe. The method described here eliminates the need for the fluorescently labeled probe, thereby reducing chemistry cost. Furthermore, technical complexity is reduced by requiring only one-color fluorescence. In our assay, we use a common unlabeled reverse primer that binds downstream of the allelic variation. Two unlabeled, allele-specific forward primers bind preferentially to either the wild-type or mutant allele at the site of allelic variation. The mutant forward primer contains a non-annealing tail (ie: stretch of the DNA primer that does not anneal to the initial DNA template) that will cause the ultimate mutant PCR product to be longer than the wild-type product. Digital PCR is characterized by dividing up a bulk PCR reaction volume into thousands of fluidically isolated partitions or droplets. In the presence of an intercalating dye, a longer amplicon will result in a higher endpoint fluorescence in that specific dPCR partition. As such, using our novel assay, dPCR partitions containing mutant versus wild-type amplicons can be distinguished by differing levels of end-point partition fluorescence. In this study we used the BRAF gene and the clinically relevant BRAF V600E mutation to benchmark this assay using a novel integrated one-step dPCR platform. This dPCR platform consists of a single, fully-integrated instrument along with a novel micro-injection molded plastic consumable providing a simple, single-step workflow. The instrument is dry and contamination-free, making it an attractive instrument for easy transition to the clinic. Parameters including thermal-cycling time and primer concentration were serially optimized for the novel integrated dPCR platform. This work highlights that we can accurately and precisely quantify samples containing varying ratios of wild-type versus V600E BRAF down to 0.1% mutant/wild-type ratio using one color of fluorescence and a novel integrated single-step walkaway dPCR workflow. Citation Format: Megan E. Dueck, Christina Wood-Bouwens, Andrew Zayac, Robert Lin, Steve Gallagher, Hanlee Ji, Paul Hung. Single-color accurate and precise quantification of wild-type versus mutant allele using a novel integrated digital PCR platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2455.

Foot-and-mouth disease virus detection on a handheld real-time polymerase chain reaction platform.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that requires rapid control. Early detection is critical but transportation of samples to laboratory delays testing. Sensitive and specific field-deployable assays are therefore desirable. Real-time reverse transcription polymerase chain reaction (RRT-PCR) and RRT-loop-mediated isothermal amplification assays for FMDV on portable platforms have been described but none of these are handheld. In this report, we have evaluated a handheld Biomeme two3™ Real-Time PCR Thermocycler (two3) as a field-deployable platform for FMDV RRT-PCR targeting the 3D gene segment. Two3's performance was compared with the laboratory-based reference assay on the ABI7500 platform. RNA extraction using a rapid Biomeme proprietary sample prep technology (M1) was compared with MagMax RNA extraction. Two3 successfully detected FMDV isolates for six serotypes (O, A, Asia 1, SAT 1, 2 and 3). Serotype C was excluded since it has not been detected in the field since 2004. The limits of detection for serial 10-fold dilutions of cell culture isolates were equal or one log different between two3 and ABI7500. Furthermore, two3 detected FMDV RNA in multiple sample types including serum, vesicular fluid, tissue suspensions, oral fluid, oral and nasal swabs. Two3 also detected FMDV RNA directly in vesicular fluid and other samples without prior RNA extraction. Comparison of the time to first detection of a positive result in serial samples in MagMax RNA extraction/ABI7500 (MgMx/ABI) system vs. M1 RNA extraction/Two3 system revealed similar or slightly better analytical sensitivity for the MgMx/ABI system. Overall, RNA extraction by M1 yielded good results and FMDV RNA detection on two3 was not significantly different from the ABI7500. Therefore, two3 could potentially enable sensitive penside detection of FMDV within an hour using M1-extracted RNA or direct testing of vesicular fluid and swabs without RNA extraction thereby ensuring prompt implementation of appropriate control measures.

AcornFusion: A novel tool to detect gene fusion events in DNA-Seq data.

e14650 Background: Gene fusions, resulting from chromosomal breakage and rearrangement, play a critical role during oncogenesis. Recently, a growing number of gene fusions are being considered as important therapeutic targets in oncology, for instance, Anaplastic lymphoma receptor tyrosine kinase (ALK) fusion, which confer exquisite sensitivity to ALK tyrosine kinase inhibitors (TKIs). However, available methods are inadequate for unbiased identification of potentially targetable fusions. Hence, we developed a novel fusion gene analyzing method named AcornFusion by DNA-seq data. Methods: The method is purely based on the mapping results of soft-clipped reads and the distribution of the positions where the candidate breakpoints locate, which is significantly different from the traditional methods with assembly of short reads and efficient way to do the detection. 309 patients with advanced non-small cell lung cancer (NSCLC) were enrolled. Formalin-fixed, paraffin embedded (FFPE) samples were used to DNA extracting and analyzed by AcornFusion method. Immunohistochemistry (IHC) and Reverse transcriptase polymerase chain reaction (RT-PCR) platform were also applied. Results: Of 309 pts, 17 patients were identified as fusion-positive cases by AcornFusion. 15 patients were EML4-ALK fusion positive and 1 patient was HIP-ALK fusion positive, which were all confirmed by IHC. 1 patient was ROS1 rearrangement positive, which was validated by Reverse transcriptase polymerase chain reaction (RT-PCR) method. Conclusions: With this advantage of high consistency with IHC and RT-PCR, AcornFusion can be a useful tool for investigating gene fusion events in clinical settings.

Genotype and Allele Frequency of P450 Oxidoreductase *28 Gene Polymorphism in South Indian Population

Objective: To establish the normative frequency of POR *28 genetic variant in South Indian population. Methods: This study is a genetic epidemiological study conducted on healthy volunteers belonging to south India. A total of 108 healthy volunteers who satisfied the eligibility criteria were included in the study. The DNA isolation was done using the QIAGEN Blood DNA isolation kit (Qiagen). The genotyping for POR *28 polymorphism was done using TaqMan genotyping master mix kits (Applied Biosystems) in real-time polymerase chain reaction platform. The data were analyzed using Fisher's exact test. P

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.

A novel helper qPCR system for platinum detection via Pt-DNA coordination

A novel real-time polymerase chain reaction (qPCR) platform for the simple and robust detection of platinum is described for the first time. Compared with conventional qPCR, a helper template, which is related to the active template for performing qPCR, was introduced in our helper qPCR system. Several guanine (G) bases were introduced in the helper template to obtain a platinum-responsive on/off switch based on G-Pt-G coordination. Because of the helper template, a slight change in platinum concentration would significantly change the signal in the qPCR. This novel helper qPCR technique easily detects platinum with high sensitivity (1 ng/mL) and selectivity over other metal ions. Therefore, it will be a promising technique for the detection of platinum in biomedical and environmental samples.

Bartonella rochalimae Detection by a Sensitive and Specific PCR Platform.

Bartonella rochalimae is an emerging zoonotic pathogen present in the United States, South America, and Europe. The molecular detection of B. rochalimae frequently relies on polymerase chain reaction (PCR) assays that target the genus Bartonella coupled with DNA sequencing for species determination. However, the presence of other Bartonella spp. in the sample being tested may result in false-negative results for B. rochalimae, especially when Sanger sequencing is used. We developed a sensitive and specific quantitative PCR platform for B. rochalimae by targeting the intergenic transcribed spacer, gltA, and rpoB genes, which are recommended for subtyping characterization. This PCR platform achieved the limit of detection between five and 10 genomic equivalents per reaction and did not amplify DNA from other Bartonella species or selected hosts. This PCR platform is a fast and cost-effective option to be used in epidemiological evaluations of reservoirs and vectors and in detecting and quantifying B. rochalimae infection in humans.

A PCR microreactor machinery with passive micropump and battery-powered heater for thermo-cycled amplifications of clinical-level and multiplexed DNA targets.

A self-contained polymerase chain reaction (PCR) platform with miniaturized power-system is introduced. Itis powered by portable lithium batteries and integrated continuous-flow PCR amplification platform. Generally speaking, traditional commercial thermal cyclers rely on external electric supply and thus they are too big in instrument size. This prevents real-timely and field testing during PCR diagnosis. The authors are introducing a continuous-flow 3D spiral microreactor for DNA amplifications and high-resolution multiplexed targets' detection by utilizing the polyvinyl chloride (PVC) tubing-polymer to fabricate the microreactor for the first time. The whole setup (that can all be placed in one hand) includes (a) the thermo-cycled control (5.5cm width, 10cm length and 11cm height), (b) the passive continuous-flow control, and (c) the trapezoidal PCR microreactor. The PCR platform can work for 4.5h continuously. With minimal accessories and operations, the total cost of the self-contained PCR machinery is <20 $, much lower than the mainstream of commercial PCR machinery. By waiving external electric supply, this miniaturized PCR platform is applied to amplify the typical DNA fragments of plasma isolated hepatitis B virus (HBV), influenza virus (H7N9avian influenza) bacterium (Escherichia coli) plasmid and multiplexed targets. The efficiency of the method is 70% of that of commercial thermal cycler (CFX Connect, Bio Rad). The DNA of H7N9avian influenza can be detected in concentrations as low as 103 copies per μL. Graphical abstract By utilizing the polyvinyl chloride (PVC) tubing-polymer to fabricate the microchip for the first time, this paper introduces a 3D spiral microreactor with a miniaturized power-system supplied by portable AA-batteries, applied in DNA amplifications and high-resolution multiplexed targets' detection. In this microdevice, we made the machinery portable by waiving the external plugs, which solved the problem of traditional commercial thermal cyclers about large volume and expensive price.

Developments in molecular diagnostics

The present article provides an overview on the rapidly evolving scenario in clinical molecular diagnostics. The concept of clinical validity and utility underpins the use of molecular diagnostic tests in the clinical laboratory, and continuous sharing of expertise between clinical and laboratory partners will enhance appropriateness of molecular testing and interpretation, thus improving personalized medicine. Thanks to the technological advances made in molecular diagnostics, above all in next generation sequencing (NGS) and digital polymerase chain reaction (dPCR) platforms, clinical laboratories benefit from rapid translation of established and emerging molecular tests. The examples of their application which will be discussed encompass cystic fibrosis (CF) (qualitative molecular) and BCR/ABL1 (quantitative molecular) tests. A final consideration on total quality assurance in molecular testing is also made and the role of accreditation is underlined as one of the main factors in guaranteeing the overall quality of molecular test results.