Showing papers on "Multiplex polymerase chain reaction published in 2020"
TL;DR: It is shown that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR in the ARTIC Network's original (V1) and modified (V3) primer set.
Abstract: Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, as sample's viral load become low, rapid decrease in abundances of several amplicons were seen. In this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex PCR. Based on this, we propose 12 alternative primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network's original (V1) and modified (V3) primer set.
145 citations
TL;DR: A design strategy for virus detection tests could lead to improved detection of SARS-CoV-2 coronavirus, and Justin Lee of the Institute for Basic Science, Daejon, Republic of Korea, and coworkers have devised a protocol for optimizing assay performance.
Abstract: SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCR-based protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.
121 citations
TL;DR: Suo et al. as mentioned in this paper explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene.
Abstract: Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.
98 citations
TL;DR: A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative).
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.
47 citations
TL;DR: The results of these experiments clearly indicate that primer dimer formation is one critical cause of coverage bias in ARTIC protocol and some of the problematic primers are detectable by observing primer dimers in raw NGS sequence reads and replacing them with alternatives as shown in this study.
Abstract: A group of biologists, ARTIC Network, has proposed a multiplexed PCR primer set for whole genome analysis of the novel corona virus, SARS-CoV-2, soon after the epidemics of this pathogen was revealed. The primer set seems to have been adapted already by many researchers worldwide and contributed for the high-quality and prompt genome epidemiology of this potential pandemic virus. We have also seen the great performance of their primer set and protocol; the primer set was able to amplify all desired PCR products with fairy small amplification bias from clinical samples with relatively high viral load. However, we observed acute drop of reads derived from two particular PCR products, 18 and 76, out of the 98 designated products as sample’s viral load decreases. We suspected the reason for this low coverage issue was due to dimer formation between primers used to amplify those two PCR products. Here, we propose replacing just one of those primers, nCoV-2019_76_RIGHT(−), to a newly designed primer. The result of the replacement of primer showed improvement in coverage at both regions targeted by the products, 18 and 76. We expect this simple modification will extend the limit for whole SARS-CoV-2 genome analysis to samples with lower viral load and enhance genomic epidemiology of this pathogen.
45 citations
TL;DR: In this paper, a novel gold nanoparticles (AuNPs)-assisted multiplex PCR assay for rapid, simple and simultaneous detection of Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7, which are the top three foodborne pathogenic bacteria.
Abstract: Foodborne pathogens are a major cause of foodborne illness, leading to a growing food safety problem in public health. This work aims to develop a novel gold nanoparticles (AuNPs)-assisted multiplex PCR assay for rapid, simple and simultaneous detection of Salmonella typhimurium (S. typhimurium), Listeria monocytogenes (L. monocytogenes) and Escherichia coli O157:H7 (E. coli O157:H7), which are the top three foodborne pathogenic bacteria. Flower-shaped AuNPs (F-AuNPs) were used as a colorimetric sensor in this assay, based on PCR product that can help improve stability of the F-AuNPs in a certain concentration of salt solution. Detection of PCR product can be directly achieved by mixing it with F-AuNPs and NaCl, and the result is visible to the naked eye. Results showed that the optimal annealing temperature was 53.1 °C to amplify the three target pathogenic strains in multiplex PCR assay, and the optimal concentrations of the primer pairs were 0.4 μM for each of L. monocytogenes and E. coli O157:H7, and 0.2 μM for S. typhimurium. The colorimetric detection limit of PCR products by F-AuNPs was 3.125 ng μL−1, and the detection time was approximately 10 min. Simultaneous detection limit of the multiplex PCR method was 10 pg μL−1 for L. monocytogenes and S. typhimurium, and 50 pg μL−1 for E. coli O157:H7. Compared with conventional multiplex PCR assay, the F-AuNPs-assisted assay is a convenient, rapid and simple visual detection method. The excellent performance of the colorimetric sensor shows potential application in on-site detection of foodborne pathogenic strains in food samples.
32 citations
TL;DR: Results of the present study indicate that the m-PCR is a potential technique for the rapid detection of foodborne bacteria for routine monitoring and risk assessment of food.
31 citations
TL;DR: OpenPrimeR, an R-based tool for evaluating and designing multiplex PCR primers, provides a functional and intuitive interface and uses either a greedy algorithm or an integer linear program to compute the minimal set of primers that performs full target coverage.
30 citations
TL;DR: In this article, the authors have determined that these dropouts were due to a dimer formation between the forward primer for amplicon 18, 18-LEFT, and the reverse primer for Amplicon 76, 76-RIGHT.
Abstract: Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, when sample’s viral load was low, several amplicons, especially amplicons 18 and 76, exhibited low coverage or complete dropout. We have determined that these dropouts were due to a dimer formation between the forward primer for amplicon 18, 18_LEFT, and the reverse primer for amplicon 76, 76_RIGHT. Replacement of 76_RIGHT with an alternatively designed primer was sufficient to produce a drastic improvement in coverage of both amplicons. Based on this result, we replaced 12 primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network’s original (V1) and modified (V3) primer set.
29 citations
TL;DR: The AusDiagnostics respiratory MT-PCR assay is a reliable assay for detection of SARS-CoV-2 and is compared with an in-house RT- PCR assay at the State Reference Laboratory.
27 citations
TL;DR: Easy-to-use Strip PCR is recommended for rapid diagnosis of infectious uveitis as its results are equivalent to that of conventional qPCR.
TL;DR: The method provided in this study can detect 11 foodborne pathogens in food, which can effectively prevent the spread of pathogenic microorganisms and be used for diagnosis to find out the cause of food poisoning incidents.
Abstract: Salmonella enterica, Listeria monocytogenes, Shigella flexneri, Escherichia coli O157:H7, Vibrio parahaemolyticus, Staphylococcus aureus, Vibrio cholerae, Clostridium botulinum type A, Bacillus cereus, Clostridium perfringens Alpha toxin, and Yersinia enterocolitica are 11 common foodborne pathogens. Traditional bacterial culture methods for detecting pathogens are time-consuming and labor-intensive. Multiplex PCR technology, which can detect multiple targets in a single tube, has been increasingly applied to microbial detection due to its high specificity, sensitivity, and fast response. This paper is to establish a multiplex PCR technology mediated by a common primer for the detection of these 11 common foodborne pathogens in order to achieve the goal of nondirectional screening for these 11 common foodborne pathogens. The specificity of the established CP-MPCR detection system was first verified by 100 clinical isolates. The sensitivity of the CP-MPCR detection system was then detected by using cultured bacteria preparations and has been confirmed with a high sensitivity of 103 to 104 CFU/mL, among them, the sensitivity of the CP-MPCR for Vibrio cholerae and S. flexneri can even achieve 102 CFU/mL. Sixty anal swab samples collected from Suzhou CDC and 16 enrichment cultured solutions of food samples collected from the Suzhou Food Inspection and Testing Center were tested using the CP-MPCR system. A total of 32 positive results were detected. PRACTICAL APPLICATION: Food poisoning incidents occur frequently around the world, mainly because of the contamination of food by pathogenic bacteria and serious harm to human health. The method provided in this study can detect 11 foodborne pathogens in food, which can effectively prevent the spread of pathogenic microorganisms. At the same time, for the food poisoning incident that has already occurred, this method can be used for diagnosis to find out the cause.
TL;DR: A multiplex-PCR-based method comprised of 172 pairs of specific primers is developed and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers, with the additional benefit of its potential for uncovering mutational diversity and novel pathogens at low sequencing depth.
Abstract: Many detection methods have been used or reported for the diagnosis and/or surveillance of SARS-CoV-2. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive, claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by RT-PCR, probably due to loss or degradation of virus RNA in the sampling process, or even mutation of the virus genome. Therefore, developing highly sensitive methods is imperative to ensure robust detection capabilities. With the goal of improving sensitivity and accommodate various application settings, we developed a multiplex-PCR-based method comprised of 172 pairs of specific primers, and demonstrate its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produces clean characteristic target peaks of defined sizes, which allows for direct identification of positives by electrophoresis. In addition, optional sequencing can provide further confirmation as well as phylogenetic information of the identified virus(es) for specific strain discrimination, which will be of paramount importance for surveillance purposes that represent a global health imperative. Finally, we also developed in parallel and tested a multiplex-PCR-based metagenomic method that is amenable to detect SARS-CoV-2, with the additional benefit of its potential for uncovering mutational diversity and novel pathogens at low sequencing depth.
TL;DR: The ability of the MIL-based dispersive liquid-liquid microextraction (DLLME) direct-multiplex-qPCR method to simultaneously achieve high enrichment factors of multiple DNA fragments from human plasma is highly promising in the field of ctDNA detection.
TL;DR: In this paper, a simple and sensitive strategy to test three foodborne pathogenic bacteria (E. coli, L. monocytogenes, and S. Typhimurium) was reported.
TL;DR: FilmArray GI panel showed a very good diagnostic performance compared to culture for diagnosis of infectious diarrhoea and gave a more detailed picture of the spectrum of the pathogens involved.
Abstract: Background: Infectious diarrhoea is a significant cause of morbidity worldwide. Culture and microscopy are time-consuming and have a low yield. New rapid molecular methods such as multiplex PCR, ha...
TL;DR: An updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis are provided and the MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material.
Abstract: DNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material. Here, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each amplicon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon. Here, we show that the MBPS assay can amplify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1–5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA. The MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.
TL;DR: The proposed high‐throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.
Abstract: Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high-throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high-throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V- and J-segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T-lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.
TL;DR: A multiplex PCR method is developed to distinguish six closely related species in the Lactobacillus acidophilus group based on species-specific primer sets, indicating that species- specific primer sets designed from the genome comparison could identify one strain within the six LactOBacillus species by a single PCR reaction.
Abstract: Many Lactobacillus species are frequently isolated from dairy products, animal guts, and the vaginas of healthy women. However, sequencing-based identification of isolated Lactobacillus strain is time/cost-consuming and lobor-intensive. In this study, we developed a multiplex PCR method to distinguish six closely related species in the Lactobacillus acidophilus group (L. gasseri, L. acidophilus, L. helveticus, L. jensenii, L. crispatus, and L. gallinarum), which is based on species-specific primer sets. Altogether, 86 genomes of 9 Lactobacillus species from the National Center of Biotechnology Information (NCBI) database were compared to detect species-specific genes and design six species-specific primer sets. The PCR conditions of the individual primer sets were optimized via gradient PCR methods. A final multiplex PCR condition was also optimized for a mixture of all six primer sets mixed. When identifying a single strain, the optimized multiplex PCR method can specifically detect one of the six species, but no band was amplified at least from the other Lactobacillus and Enterococcus species. These results indicated that species-specific primer sets designed from the genome comparison could identify one strain within the six Lactobacillus species by a single PCR reaction. Using the method described here, we will be able to save time, cost, and labor during species identification and screening of commercially important probiotic lactobacilli.
TL;DR: A SVV-1 real-time RT-PCR assay that has two targets, one in the 5' untranslated region (5'UTR, this study) and the other in the 3D region (3D) region was developed and multiplexed with published FMDV assays that specifically detected the target viruses without cross reacting to SVv-1 or to other common porcine viruses.
Abstract: Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically important vesicular disease in cloven-hoofed animals that is clinically indistinguishable from symptoms caused by Seneca Valley virus 1 (SVV-1). To differentiate SVV-1 from FMDV infections, we developed a SVV-1 real-time RT-PCR (RT-qPCR) assay and multiplexed with published FMDV assays. Two published FMDV assays (Journal of the American Veterinary Medical Association, 220, 2002, 1636; Journal of Virological Methods, 236, 2016, 258) targeting the 3D polymerase (3D) region were selected and multiplexed with the SVV-1 assay that has two targets, one in the 5' untranslated region (5' UTR, this study) and the other in the 3D region (Journal of Virological Methods, 239, 2017, 34). In silico analysis showed that the primers and probes of SVV-1 assay matched 98.3% of the strain sequences (113/115). The primer and probe sequences of the Shi FMDV assay matched 85.4% (806/944), and that of the Callahan FMDV assay matched 62.7% (592/944) of the sequences. The limit of detection (LOD) for the two multiplex RT-qPCR assays for SVV-1 was both 9 copies per reaction by cloned positive plasmids and 0.16 TCID50 per reaction by cell culture. The LOD for FMDV by both multiplex assays was 11 copies per reaction using cloned positive plasmids. With cell cultures of the seven serotypes of FMDV, the Shi assay (Journal of Virological Methods, 236, 2016, 258) had LODs between 0.04 and 0.18 TCID50 per reaction that were either the same or lower than the Callahan assay. Interestingly, multiplexing with SVV-1 increased the amplification efficiencies of the Callahan assay (Journal of the American Veterinary Medical Association, 220, 2002, 1636) from 51.5%-66.7% to 89.5%-96.6%. Both assays specifically detected the target viruses without cross-reacting to SVV-1 or to other common porcine viruses. An 18S rRNA housekeeping gene that was amplified from multiple cloven-hoofed animal species was used as an internal control. The prevalence study did not detect any FMDV, but SVV-1 was detected from multiple types of swine samples with an overall positive rate of 10.5% for non-serum samples.
TL;DR: The mPCR assay was sensitive and specific, and the amplified product could be analyzed easily, and could be used effectively to identify the suspected colonies of DEC in the primary culture of the specimen.
TL;DR: This study provides a fast and simple detection method for regulating labeling of animal-derived ingredients in meat products and its results were consistent with their labeled meat species.
Abstract: The continuous development of fast and simple new methods to identify animal-derived ingredients is very important for the authentication of meat products. This study intended to develop a multiplex PCR method using new species-specific nuclear DNA (nDNA) sequences for the detection of ingredients derived from sheep/goat, bovine, chicken, duck and pig in meat products. Sequence alignment analysis in 53 species showed high specificity of species-specific nDNA. Species-specific primers were designed on the conservative region of each species-specific nDNA sequence. The specificity and conservation of the sequences and primers were verified by PCR reaction and sequencing with the limit of detection down to 0.5 ng. Then, a species-specific multiplex PCR method was developed and optimized to simultaneously detect sheep/goat (237 bp), bovine (223 bp), chicken (192 bp), duck (168 bp) and pig (154 bp) in one reaction. Various processed meat products containing one or more animal-derived ingredients were detected by the developed multiplex PCR method, and the results were consistent with their labeled meat species. Our study provides a fast and simple detection method for regulating labeling of animal-derived ingredients in meat products.
TL;DR: This PCR assay will compensate for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, among others, in clinical and public health settings.
Abstract: A multiplex PCR assay in a single tube was developed for the detection of the carbapenemase genes of Enterobacteriaceae. Primers were designed to amplify the following six carbapenemase genes: blaKPC, blaIMP, blaNDM, blaVIM, blaOXA-48-like, and blaGES. Of 70 blaIMP variants, 67 subtypes were simulated to be PCR-positive based on in silico simulation and the primer-design strategy. After determining the optimal PCR conditions and performing in vitro assays, the performance of the PCR assay was evaluated using 51 and 91 clinical isolates with and without carbapenemase genes, respectively. In conclusion, the combination of multiplex PCR primers and QIAGEN Multiplex PCR Plus Kit was used to determine the best performance for the rapid and efficient screening of carbapenemase genes in Enterobacteriaceae. The assay had an overall sensitivity and specificity of 100%. This PCR assay compensates for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, in clinical and public health settings.
TL;DR: The principles of PCR-based assays that are commonly used in transfusion medicine are described, popular because they analyze multiple nucleotides in a single assay and have a high-throughput potential.
Abstract: DNA-based assays are powerful tools to predict the blood group of an individual and are rapidly gaining in popularity. DNA, which can be extracted from various sources using commercial kits, is amplified by PCR to obtain a sufficient amount of the target of interest for analysis. There are different types of PCR assays: standard single PCR (followed by RFLP or sequencing), allele-specific PCR, multiplex PCR, and real-time PCR. Microarray platforms are a newer application of molecular testing, popular because they analyze multiple nucleotides in a single assay and have a high-throughput potential. This review briefly describes the principles of PCR-based assays that are commonly used in transfusion medicine.
TL;DR: In this paper, a multiplex-PCR-based method comprised of 172 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers.
Abstract: Many detection methods have been used or reported for the diagnosis and/or surveillance of SARS-CoV-2. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive, claiming detection of about 5 copies of viruses. However, it has been reported that only 47-59% of the positive cases were identified by RT-PCR, probably due to loss or degradation of virus RNA in the sampling process, or even mutation of the virus genome. Therefore, developing highly sensitive methods is imperative to ensure robust detection capabilities. With the goal of improving sensitivity and accommodate various application settings, we developed a multiplex-PCR-based method comprised of 172 pairs of specific primers, and demonstrated its efficiency to detect SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. In addition, optional sequencing can provide further confirmation as well as phylogenetic information of the identified virus(es) for specific strain discrimination, which will be of paramount importance for surveillance purposes that represent a global health imperative. Finally, we also developed in parallel a multiplex-PCR-based metagenomic method that is amenable to detect SARS-CoV-2, with the additional benefit of its potential for uncovering mutational diversity and novel pathogens at low sequencing depth.
TL;DR: The multiplex PCR results showed that serovar 4h strains could be specifically identified from all tested strains, including various L. monocytogenes serovars, Listeria spp.
Abstract: Listeria monocytogenes (L monocytogenes) is a ubiquitous foodborne pathogen that comprises 14 serotypes, of which serovar 4h is a novel serotype recently reported Serovar 4h L monocytogenes belonging to hybrid sub-lineage II exhibit hypervirulent features Conventional biochemical tests and widely used PCR-based serogrouping schemes could not distinguish serovar 4h strains In this study, we developed a new multiplex PCR assay for rapid detection of serotype 4h L monocytogenes Three primer pairs based on the target genes, LMxysn_1095, lmo1083, and smcL, were designed The multiplex PCR results showed that serovar 4h strains could be specifically identified from all tested strains, including various L monocytogenes serovars, Listeria spp, and other species The detection limits of the multiplex PCR were 291 fg/μL for genomic DNA and 55 × 106 CFU/mL for bacterial suspension Furthermore, pork meat artificially contaminated with serovar 4h L monocytogenes in a concentration of 18 × 103-18 × 100 CFU/10 g were successfully detected within 10-16 h These results demonstrate that the multiplex PCR with high specificity and sensitivity is applicable for the rapid detection of L monocytogenes serotype 4h strains
TL;DR: The study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites.
Abstract: A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.
TL;DR: Analysis of Pseudomonas aeruginosa isolates for theirClonality, virulence factors, and resistance characteristics is achievable by combining the clonality evaluation of the core genome based on multiple‐locus targeting methods with other methods that can identify specific virulence and antimicrobial genes.
Abstract: In recent years, numerous outbreaks of multidrug-resistant Pseudomonas aeruginosa have been reported across the world. Once an outbreak occurs, besides routinely testing isolates for susceptibility to antimicrobials, it is required to check their virulence genotypes and clonality profiles. Replacing pulsed-field gel electrophoresis DNA fingerprinting are faster, easier-to-use, and less expensive polymerase chain reaction (PCR)-based methods for characterizing hospital isolates. P. aeruginosa possesses a mosaic genome structure and a highly conserved core genome displaying low sequence diversity and a highly variable accessory genome that communicates with other Pseudomonas species via horizontal gene transfer. Multiple-locus variable-number tandem-repeat analysis and multilocus sequence typing methods allow for phylogenetic analysis of isolates by PCR amplification of target genes with the support of Internet-based services. The target genes located in the core genome regions usually contain low-frequency mutations, allowing the resulting phylogenetic trees to infer evolutionary processes. The multiplex PCR-based open reading frame typing (POT) method, integron PCR, and exoenzyme genotyping can determine a genotype by PCR amplifying a specific insertion gene in the accessory genome region using a single or a multiple primer set. Thus, analyzing P. aeruginosa isolates for their clonality, virulence factors, and resistance characteristics is achievable by combining the clonality evaluation of the core genome based on multiple-locus targeting methods with other methods that can identify specific virulence and antimicrobial genes. Software packages such as eBURST, R, and Dendroscope, which are powerful tools for phylogenetic analyses, enable researchers and clinicians to visualize clonality associations in clinical isolates.
TL;DR: The LAMP assay was developed for rapid, simple, effective species identification of Mythimna loreyi by analyzing the mitochondrial genome, which found the species-specific sequence was found at the coding region of the NADH dehydrogenase subunit 5 gene.
Abstract: The Mythimna loreyi (Duponchel) is one of the well-known invasive noctuid pests in Africa, Australia, and many Asian countries. However, it is difficult to identify the invasive and morphologically similar species, Mythimna separate, which occur at the cornfield in the larvae stage. Currently, the molecular biology method for diagnosing M. loreyi species is only using the mtCO1 universal primer (LCO1490, HCO2198), which requires a lot of time and effort, such as DNA extraction, PCR, electrophoresis, and sequencing. In this study, the LAMP assay was developed for rapid, simple, effective species identification. By analyzing the mitochondrial genome, the species-specific sequence was found at the coding region of the NADH dehydrogenase subunit 5 gene. Based on this unique sequence, four LAMP primers and two loop primers were designed. The F3 and B3 primers were able to diagnose species-specific, in general, and multiplex PCR and specifically reacted within the inner primers in LAMP assay. The optimal incubation condition of the LAMP assay was 61 °C for 60 min with four LAMP primers, though additional loop primers, BF and LF, did not significantly shorten the amplification time. The broad range of DNA concentration was workable in LAMP assay, in which the minimum detectable DNA concentration was 100 pg. DNA releasing method was applied, which took five minutes of incubation at 95 °C without the DNA extraction process. Only some pieces of tissue of larvae and adult samples were needed to extract DNA. The incidence of invasive pests is gradually diversifying. Therefore, this simple and accurate LAMP assay is possibly applied in the intensive field monitoring for invasive pests and integrated management of Mythimna loreyi.
TL;DR: The multiplex PCR-based assay had a higher sensitivity and specificity than conventional microbiology when only the pathogens on the pneumonia panel were taken into account.
Abstract: The identification of the pathogens in pleural effusion has mainly relied on conventional bacterial culture or single species polymerase chain reaction (PCR), both with relatively low sensitivity. We investigated the efficacy of a commercially available multiplex bacterial PCR assay developed for pneumonia to identify the pathogens involved in pleural infection, particularly empyema. A prospective, monocentric, observational study including 194 patients with pleural effusion. Patients were evaluated based on imaging, laboratory values, pleura ultrasound and results of thoracentesis including conventional microbiology studies during hospitalisation. Multiplex bacterial PCR (Curetis Unyvero p55) was performed in batch and had no influence on therapeutic decisions. Overall, there were 51/197 cases with transudate and 146/197 with exudate. In 42% (n = 90/214) there was a clinical suspicion of parapneumonic effusion and the final clinical diagnosis of empyema was made in 29% (n = 61/214) of all cases. The most common microorganisms identified in the cases diagnosed with empyema were anaerobes [31] followed by gram-positive cocci [10] and gram-negative rods [4]. The multiplex PCR assay identified more of the pathogens on the panel than the conventional methods (23.3% (7/30) vs. 6.7% (2/30), p = 0.008). The multiplex PCR-based assay had a higher sensitivity and specificity than conventional microbiology when only the pathogens on the pneumonia panel were taken into account. A dedicated pleural empyema multiplex PCR panel including anaerobes would be needed to cover most common pathogens involved in pleural infection.