Polymerase Chain Reaction Probes Research Articles

Monoallelic pathogenic IFT140 variants are a common cause of autosomal dominant polycystic kidney disease-spectrum phenotype.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder, characterized by development and enlargement of kidney cysts, eventually leading to end-stage kidney disease (ESKD). Pathogenic variants in the PKD1 and PKD2 genes are the major cause of ADPKD; additional rare variants in the GANAB, DNAJB11, ALG5 and ALG9 genes have been found in a minority of ADPKD patients. More recently, a significant number of ADPKD families have been linked to monoallelic variants in the IFT140 gene. In this retrospective study, we tested the prevalence of the known causative genes of ADPKD-spectrum phenotype, including the PKD1, PKD2, GANAB, DNAJB11, ALG5, ALG and IFT140 genes, in a cohort of 129 ADPKD patients who consecutively underwent genetic testing in a single centre in Italy. Genetic testing utilized a combination of targeted next-generation sequencing, long-range polymerase chain reaction, Sanger sequencing and multiplex ligation-dependent probe amplification. Clinical evaluation was conducted through renal function testing and imaging features, including ultrasonography, computer tomography and magnetic resonance imaging. Of the 129 enrolled patients, 86 (66.7%) had pathogenic variants in PKD1 and 28 (21.7%) in PKD2, loss of function pathogenic variants in the IFT140 gene were found in 3 unrelated patients (2.3%), no pathogenic variants were found in other ADPKD genes and 12 patients (9.3%) remained genetically unresolved (ADPKD-GUR). Familial clinical and genetic screening of the index patients with ADPKD due to an IFT140 pathogenic variant (ADPKD-IFT140) allowed identification of eight additional affected relatives. In the 11 ADPKD-IFT140 patients, the renal phenotype was characterized by mild and late-onset PKD, with large renal cysts and limited kidney insufficiency. Extrarenal manifestations, including liver cysts, were rarely seen. Our data suggest the monoallelic pathogenic IFT140 variants are the third most common cause of the ADPKD-spectrum phenotype in Italy, usually associated with a mild and atypical renal cystic disease.

Coronary atherosclerosis and periodontitis have similarities in their clinical presentation.

Periodontitis (PE) and coronary heart disease (CHD) possess multiple mechanisms for a putative association. This case-control study compared the periodontal status among CHD subjects to controls without CHD, while also investigating atheroma invasion by known periodontal pathogens. 161 subjects participated in this study were divided into three CHD groups: No CHD, chronic CHD, acute CHD. Additional analysis involved grouping subjects according to number of atheromas: no atheroma, 1-4 atheromas, 5-18 atheromas. Data were collected from medical records, periodontal examinations, and questionnaires that included demographic, behavioral, and oral health variables. Angiographic catheterizations were analyzed according to the number of atheroma lesions, lesion size, lesion location, and atheroma lesion stability. Lipoprotein profile, inflammatory markers and cells were analyzed. The microbiological branch added 30 individuals who had their atheroma lesion and subgingival plaque analyzed using polymerase chain reaction probes against the 16 s region, red complex and Aggregatibacter actinomycetemcomitans' DNA. Subjects with CHD had high levels of systemic inflammatory markers and low levels of high-density lipoproteins compared to subjects without CHD. Subjects without CHD and clear coronaries had a prevalence of mild CAL, while individuals with more atheroma lesions had advanced CAL and more active PE. Subjects with more advanced CAL were 4 times more likely to have CHD compared to subjects with less, which is comparable to smoking. Only 4 subjects had the screened pathogens detected in atheroma, although these subjects also have the screened pathogens in subgingival plaque. However, 80% of atheromas had bacteria. CHD and PE showed similarities in progression while active PE led to more atheroma lesions that also tended to be larger in size.

Early Detection of the Emerging SARS-CoV-2 BA.2.86 Lineage Through Wastewater Surveillance Using a Mediator Probe PCR Assay - Shenzhen City, Guangdong Province, China, 2023.

The emergence of the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineage, BA.2.86, has sparked global public health concerns for its potential heightened transmissibility and immune evasion. Utilizing data from Shenzhen's city-wide wastewater surveillance system, we highlight the presence of the BA.2.86 lineage in Shenzhen. A mediator probe polymerase chain reaction (PCR) assay was developed to detect the BA.2.86 lineage in wastewater by targeting a specific mutation (Spike: A264D). Between September 19 and December 10, 2023, 781 wastewater samples from 38 wastewater treatment plants (WWTPs) and 9 pump stations in ten districts of Shenzhen were examined. Through multiple short-amplicon sequencing, three positive samples were identified. The BA.2.86 lineage was identified in the wastewater of Futian and Nanshan districts in Shenzhen on December 2, 2023. From December 2 to 10, a total of 21 BA.2.86-positive wastewater samples were found across 6 districts (Futian, Nanshan, Longhua, Baoan, Longgang, and Luohu) in Shenzhen. The weighted average viral load of the BA.2.86 lineage in Shenzhen's wastewater was 43.5 copies/L on December 2, increased to 219.8 copies/L on December 4, and then decreased to approximately 100 copies/L on December 6, 8, and 10. The mediator probe PCR assay, designed for swift detection of low viral concentrations of the BA.2.86 lineage in wastewater samples, shows promise for detecting different SARS-CoV-2 variants. Wastewater surveillance could serve as an early detection system for promptly identifying specific SARS-CoV-2 variants as they emerge.

Clinical Implementation of β-Tubulin Gene-Based Aspergillus Polymerase Chain Reaction for Enhanced Aspergillus Diagnosis in Patients with Hematologic Diseases: A Prospective Observational Study.

The β-tubulin (benA) gene is a promising target for the identification of Aspergillus species. Assessment of the clinical implementation and performance of benA gene-based Aspergillus polymerase chain reaction (PCR) remains warranted. In this study, we assessed the analytical performance of the BenA probe PCR in comparison with the Aspergenius kit. We prospectively collected bronchoalveolar lavage (BAL) fluid via diagnostic bronchoscopy from adult patients with hematologic diseases. BenA gene-based multiplex real-time PCR and sequential melting temperature analysis were performed to detect the azole resistance of Aspergillus fumigatus. In total, 76 BAL fluids in 75 patients suspicious of invasive pulmonary aspergillosis (IPA) were collected. Before the application of PCR, the prevalence of proven and probable IPA was 32.9%. However, after implementing the benA gene-based PCR, 15.8% (12 out of 76) of potential IPA cases were reclassified as probable IPA. The analytical performance of the BenA probe PCR in BAL samples was comparable to that of the Aspergenius kit. The diagnostic performance was as follows: sensitivity, 52.0%; specificity, 64.7%; positive predictive value, 41.9%; negative predictive value, 73.3%; positive likelihood ratio, 1.473; and negative likelihood ratio, 0.741. Moreover, benA gene-based Aspergillus PCR discriminated all major sections of Aspergillus, including cryptic species such as Aspergillus tubingensis. Sequential melting temperature analysis successfully detected 2 isolates (15.4%) of A. fumigatus carrying resistant mutations. BenA gene-based Aspergillus PCR with melting temperature analysis enhances diagnostic accuracy and detects not only cryptic species but also resistant mutations of A. fumigatus. It shows promise for clinical applications in the diagnosis of IPA.

Genetic Factors of Teeth Impaction: Polymorphic and Haplotype Variants of PAX9, MSX1, AXIN2, and IRF6 Genes.

In recent research, there has been a growing awareness of the role of genetic factors in the positioning and eruption of teeth in the maxilla and mandible. This study aimed to evaluate the potential of specific polymorphic markers of single nucleotide polymorphisms (SNPs) located within the PAX9, MSX1, AXIN2, and IRF6 genes to determine the predisposition to tooth impaction. The study participants were divided into two groups: the first group consisted of individuals with at least one impacted secondary tooth. In contrast, the second group (control group) had no impacted teeth in their jaws. To analyze the genes, real-time PCR (polymerase chain reaction) and TaqMan probes were utilized to detect the selected polymorphisms. The findings suggest that disruptions in the structure and function of the mentioned genetic factors such as polymorphic and haplotype variants of PAX9, MSX1, AXIN2, and IRF6 genes, which play a direct role in tooth and periodontal tissue development, might be significant factors in tooth impaction in individuals with genetic variations. Therefore, it is reasonable to hypothesize that tooth impaction may be influenced, at least in part, by the presence of specific genetic markers, including different allelic variants of the PAX9, AXIN2, and IRF6 genes, and especially MSX1.

Nasal discharge: A unique enhanced alternative for the detection of respiratory pathogens in adults.

Upper respiratory tract infections are a significant cause of social- and disease burden worldwide. Currently, invasive and uncomfortable molecular detection methods are used for respiratory pathogen detection. We aimed to assess the ability and bearability of a rhinorrhea swab (RS) to detect respiratory pathogens in comparison to the combined nasopharyngeal and oropharyngeal swab (NP/OP). This study was performed at a Public Health Service severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) testing facility between November and December 2022 in the Netherlands. Adults aged 16 years and older, being subjected to a standard of care NP/OP swab with nasal discharge, were included and received an additional RS. Respiratory pathogen detection was evaluated using SARS-CoV-2 polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) PCR. Bearability was evaluated using visual analog scale (VAS) scores and a questionnaire. A total of 100 adults with a mean age ± SD of 46 ± 16 years were included. The NP/OP swab detected 104 pathogens, the RS 83 pathogens (p < 0.001), and in total 108 respiratory pathogens were identified in 89 adults (89%). The ability to detect respiratory pathogens compared between the RS and the combined NP/OP swab revealed a sensitivity of 82% (95% CI 73%-89%) and specificity of 100% (95% CI 72%-100%). RS were significantly more bearable than the combined NP/OP swab (p value < 0.001). Therefore, nasal discharge found in adults can be used as an adequate reliable medium for respiratory pathogen detection using SARS-CoV-2 PCR and MLPA PCR.

Development of a novel ddPCR assay for nonclinical and clinical pharmacokinetic characterization of ICVB-1042.

e14693 Background: ICVB-1042 is a novel chimeric oncolytic Adenovirus (Ad) with potential for intravenous administration. A key component of the clinical assessment of ICVB-1042 will be pharmacokinetic (PK) viral genome analysis by Polymerase Chain Reaction (PCR) to understand plasma viral kinetics and tumor replication. Droplet Digital PCR (ddPCR) is more sensitive and more accurately quantifies a lower template concentration compared to traditional quantitative PCR. Our ddPCR assay targets ICVB-1042 and will not amplify other Ad5 or Ad34 viruses. The aim is to show that this assay specifically measures ICVB-1042 and may be used as a clinical biomarker. Methods: PCR primers and probe were designed to target the Ad5/Ad34 chimeric fiber of ICVB-1042. Template ICVB-1042 was spiked into representative human and murine sample matrices. Preclinical studies tested assay performance in vitro using patient derived disassociated tumor cells (DTCs) and in vivo using tumor bearing and non-tumor bearing mouse models. Viral replication and PK were assessed in vitro by analyzing supernatant of infected cells at multiple timepoints and in vivo with serial bleeds from different animals. Viral distribution was assessed by isolating viral DNA from homogenized tissue samples taken at multiple timepoints. Results: Analysis of ICVB-1042 by ddPCR showed linear quantification over a wide range of viral concentrations (5E5-5E12 viral genomes/mL) with sensitivity to low concentrations of virus (lower limit of quantification = 1.11E2 total viral genomes). Viral genomes were detected and quantified in human plasma samples spiked with ICVB-1042. Non-spiked samples were negative for signal. ICVB-1042 viral genomes were also detectible in tumor sections from ICVB-1042-treated NSG mice. Increasing viral genomes could be detected in vitro at days 6 and 9 post ICVB-1042 infection in the supernatants of several DTCs (2 donors each). The measured increase in viral genomes was accompanied by viral mediated cell death. ICVB-1042 could be detected in vivo in the plasma of non-tumor bearing CD-1 mice 1 and 24 hours post ICVB-1042 dosing but was cleared and not detected out to 90 days post dose. ICVB-1042 was detected post dose in the plasma of tumor bearing NSG mice and was again cleared. ICVB-1042 was detectable in plasma samples and accompanied by anti-tumor efficacy at later timepoints. Studies directly comparing tumor bearing to non-tumor bearing NSG mice have shown that the virus present in the plasma is due to tumor specific replication. Tissue viral genome distribution differences could also be detected in C57BL/6 mice expressing huCD46, the viral tropism of ICVB-1042. Conclusions: ICVB-1042 viral genomes can be detected using ddPCR. This assay can be used to assess viral replication in vitro and PK and distribution in vivo. Use of ddPCR to detect ICVB-1042 in patient samples will be key to the characterization of clinical efficacy.

Validation of a rapid HLA-DQA1*05 pharmacogenomics assay to identify at-risk resistance to anti-tumor necrosis factor therapy among patients with inflammatory bowel disease.

The HLA-DQA1*05 variant (rs2097432) is associated with increased risk of immunogenicity to tumor necrosis factor antagonists, with subsequent resistance to therapy in patients with inflammatory bowel disease. Identification of these patients would optimize personalized therapeutic selection. Genomic DNA was extracted from 80 deidentified samples in an unselected patient population with an unknown rs2097432 genotype. Split sample analysis was performed using a reference laboratory. Primer probes for a TaqMan quantitative polymerase chain reaction (qPCR) assay (Thermo Fisher Scientific) were custom designed. Synthesized genomic-block fragments were used as controls. All qPCR reactions were performed using a TaqMan GTXpress Master Mix (Thermo Fisher Scientific) on the Applied Biosystems 7500 system under fast cycling conditions. Of 80 samples, 50% were wild-type reference genotypes, 22.5% were heterozygous, and 27.5% were homozygous variant calls, comparable to population data. Split analysis samples between 2 independent laboratories were 100% concordant. The detection limit tested across genomic-block controls processed in duplicate was reproducible on sample input from 10 ng titrated down to 1.25 ng across 2 independent runs. Further, analytical specificity assessed with previous wild-type reference and homozygous variant DNA spiked into genomic-block controls produced appropriate heterozygous genotypes. Here we present validation of a lab-developed test for a rapid HLA-DQA1*05 (rs2097432) pharmacogenomics assay targeting a hotspot identified by genome-wide association studies. Targeted genotyping employed here will allow for expeditious personalized therapeutic selection.

Detection of Clostridioides difficile toxin B gene in clinical stool specimens using rapid diagnostic quenching probe-polymerase chain reaction assay

We tested the accuracy of quenching probe-polymerase chain reaction (QP-PCR) for detecting Clostridioides difficile toxin B gene (tcdB) in stools from inpatients with suspected C. difficile infection and compared the results with other nucleic acid amplification tests (NAATs). Toxigenic culture results were used as reference for comparison. QP-PCR had comparable diagnostic accuracy with other NAATs and prior bead-beating enabled detection of tcdB in specimens judged as negative, without bead-beating. Taken together, the QP-PCR either with or without bead-beating showed sufficient effectiveness for detecting tcdB in stool specimens.

The genetic landscape of autosomal dominant polycystic kidney disease in Kuwait.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common renal monogenic disease, characterized by bilateral accumulation of renal fluid-filled cysts leading to progressive renal volume enlargement and gradual impairment of kidney function, often resulting in end-stage renal disease. Kuwait could provide valuable genetic insights about ADPKD, including intrafamilial phenotypic variation, given its large household size. This study aims to provide a comprehensive description of the pathogenic variants linked to ADPKD in the Kuwaiti population using multiple genetic analysis modalities and to describe and analyse the ADPKD phenotypic spectrum in terms of kidney function, kidney volume and renal survival. A total of 126 ADPKD patients from 11 multiplex families and 25 singletons were recruited into the study. A combination of targeted next-generation sequencing (tNGS), long-range polymerase chain reaction, Sanger sequencing and multiplex ligation-dependent probe amplification were utilized for genetic diagnosis. Clinical evaluation was conducted through renal function testing and ultrasonographic kidney volume analysis. We identified 29 ADPKD pathogenic mutations from 36 families achieving an overall molecular genetic diagnostic rate of 112/126 (88.9%), including 29/36 (80.6%) in families. A total of 28/36 (77.8%) families had pathogenic mutations in PKD1, of which 17/28 (60.7%) were truncating, and 1/36 (2.8%) had a pathogenic variant in the IFT140 gene. A total of 20/29 (69%) of the identified ADPKD mutations were novel and described for the first time, including a TSC2-PKD1 contiguous syndrome. Clinical analysis indicated that genetically unresolved ADPKD cases had no apparent association between kidney volume and age. We describe for the first time the genetic landscape of ADPKD in Kuwait. The observed genetic heterogeneity underlining ADPKD along with the wide phenotypic spectrum reveal the level of complexity in disease pathophysiology. ADPKD genetic testing could improve the care of patients through improved disease prognostication, guided treatment and genetic counselling. However, to fulfil the potential of genetic testing, it is important to overcome the hurdle of genetically unresolved ADPKD cases.

Rapid Diagnostic Testing for Response to the Monkeypox Outbreak - Laboratory Response Network, United States, May 17-June 30, 2022.

As part of public health preparedness for infectious disease threats, CDC collaborates with other U.S. public health officials to ensure that the Laboratory Response Network (LRN) has diagnostic tools to detect Orthopoxviruses, the genus that includes Variola virus, the causative agent of smallpox. LRN is a network of state and local public health, federal, U.S. Department of Defense (DOD), veterinary, food, and environmental testing laboratories. CDC developed, and the Food and Drug Administration (FDA) granted 510(k) clearance* for the Non-variola Orthopoxvirus Real-time PCR Primer and Probe Set (non-variola Orthopoxvirus [NVO] assay), a polymerase chain reaction (PCR) diagnostic test to detect NVO. On May 17, 2022, CDC was contacted by the Massachusetts Department of Public Health (DPH) regarding a suspected case of monkeypox, a disease caused by the Orthopoxvirus Monkeypox virus. Specimens were collected and tested by the Massachusetts DPH public health laboratory with LRN testing capability using the NVO assay. Nationwide, 68 LRN laboratories had capacity to test approximately 8,000 NVO tests per week during June. During May 17-June 30, LRN laboratories tested 2,009 specimens from suspected monkeypox cases. Among those, 730 (36.3%) specimens from 395 patients were positive for NVO. NVO-positive specimens from 159 persons were confirmed by CDC to be monkeypox; final characterization is pending for 236. Prompt identification of persons with infection allowed rapid response to the outbreak, including isolation and treatment of patients, administration of vaccines, and other public health action. To further facilitate access to testing and increase convenience for providers and patients by using existing provider-laboratory relationships, CDC and LRN are supporting five large commercial laboratories with a national footprint (Aegis Science, LabCorp, Mayo Clinic Laboratories, Quest Diagnostics, and Sonic Healthcare) to establish NVO testing capacity of 10,000 specimens per week per laboratory. On July 6, 2022, the first commercial laboratory began accepting specimens for NVO testing based on clinician orders.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

Detection of SARS-CoV-2 Reinfections by Rapid Inexpensive Methods

New SARS-CoV-2 infections are difficult to beverified, whether they are reinfections or persistent infections. The most prominent factors used for differentiating reinfections from persistent infections are whole-genome sequencing and phylogenetic analyses that require time and funds, which may not be feasible in most developing countries. This study explores reinfections with COVID-19 that harbors D614G and N501Y mutations by rapid inexpensive methods. It exploits the previously developed rapid economic methods that identified both D614G and N501Y mutations in clinical samples using real-time reverse transcriptase polymerase chain reaction (rRT-PCR) probes and conventional PCR specific primers. In the present study, an immunocompetent patient has been found with a SARS-CoV-2 N501Y reinfection without comorbidities. According to the obtained results, this study suggests that the initial infection was due to a variant that contained only D614G mutation whereas the reinfection was potentially a result of alpha variant contained three mutations confirmed by DNA sequencing, including D614G, N501Y, and A570D mutations. These techniques will support rapid detection of SARS-CoV-2 reinfections through the identification of common spike mutations in the developing countries where sequencing tools are unavailable. Furthermore, seven cases of reinfections were also confirmed by these methods. These rapid methods can also be applied to large samples of reinfections that may increase our understanding epidemiology of the pandemic.

Development of a Triplex Real-Time PCR Assay to Detect Echinococcus Species in Canid Fecal Samples

Echinococcosis is a zoonotic disease with great significance to public health, and appropriate detection and control strategies should be adopted to mitigate its impact. Most cases of echinococcosis are believed to be transmitted by the consumption of food and/or water contaminated with canid stool containing Echinococcus spp. eggs. Studies assessing Echinococcus multilocularis, Echinococcus granulosus sensu stricto, and Echinococcus shiquicus coinfection from contaminated water-derived, soil-derived, and food-borne samples are scarce, which may be due to the lack of optimized laboratory detection methods. The present study aimed to develop and evaluate a novel triplex TaqMan-minor groove binder probe for real-time polymerase chain reaction (rtPCR) to simultaneously detect the 3 Echinococcus spp. mentioned above from canid fecal samples in the Qinghai-Tibetan Plateau area (QTPA). The efficiency and linearity of each signal channel in the triplex rtPCR assay were within acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.32 cycle threshold), and the limit of detection was estimated to be 10 copies plasmid/μl reaction. In summary, the evaluation of the present method shows that the newly developed triplex rtPCR assay is a highly specific, precise, consistent, and stable method that could be used in epidemiological investigations of echinococcosis.

A multiplex real-time PCR assay for differential identification of avian Chlamydia

ABSTRACT Avian chlamydiosis is an acute or chronic disease of birds after infection by Chlamydia. Although Chlamydia psittaci is the primary agent of the disease, two additional species, Chlamydia avium and Chlamydia gallinacea, have also been recognized as potential disease agents. Therefore, the diagnosis of avian chlamydiosis requires differential identification of these avian Chlamydia species. The objective of the present study was to develop a multiplex real-time polymerase chain reaction (PCR) assay to rapidly differentiate between these three species of avian Chlamydia (C. psittaci, C. avium, and C. gallinacea) as well as to detect the genus Chlamydia. Specific genetic regions of the three species were identified by comparative analysis of their genome sequences. Also, the genus-specific region was selected based on 23S rRNA sequences. PCR primers and probes specific to the genus and each species were designed and integrated in the multiplex real-time PCR assay. The assay was highly efficient (94.8–100.7%). It could detect fewer than 10 copies of each target sequence of the genus and each species. Twenty-five Chlamydia control and field DNA samples were differentially identified while 20 other bacterial strains comprising 10 bacterial genera were negative in the assay. This assay allows rapid, sensitive, and specific detection of the genus and the three species of avian Chlamydia in a single protocol that is suitable for routine diagnostic purposes in avian diagnostic laboratories.

Development of Multiplex PCR Coupled DNA Chip Technology for Assessment of Endogenous and Exogenous Allergens in GM Soybean.

Allergenicity assessment of transgenic plants and foods is important for food safety, labeling regulations, and health protection. The aim of this study was to develop an effective multi-allergen diagnostic approach for transgenic soybean assessment. For this purpose, multiplex polymerase chain reaction (PCR) coupled with DNA chip technology was employed. The study was focused on the herbicide-resistant Roundup Ready soya (RRS) using a set of certified reference materials consisting of 0, 0.1%, 0.5%, and 10% RRS. Technically, the procedure included design of PCR primers and probes; genomic DNA extraction; development of uniplex and multiplex PCR systems; DNA analysis by agarose gel electrophoresis; microarray development, hybridization, and scanning. The use of the asymmetric multiplex PCR method is shown to be very efficient for DNA hybridization with biochip probes. We demonstrate that newly developed fourplex PCR methods coupled with DNA-biochips enable simultaneous identification of three major endogenous allergens, namely, Gly m Bd 28K, Gly m Bd 30K, and lectin, as well as exogenous 5-enolppyruvyl shikimate-phosphate synthase (epsps) expressed in herbicide-resistant roundup ready GMOs. The approach developed in this study can be used for accurate, cheap, and fast testing of food allergens.

LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species.

Background: Laboratory diagnosis of Lyme borreliosis refers to some methods with known limitations. Molecular diagnostics using specific nucleic acid probes may overcome some of these limitations. Methods: We describe the novel reporter fluorescence real-time polymerase chain reaction (PCR) probe system LoopTag for detection of Borrelia species. Advantages of the LoopTag system include having cheap conventional fluorescence dyes, easy primer design, no restrictions for PCR product lengths, robustness, high sequence specificity, applicability for multiplex real-time PCRs, melting curve analysis (single nucleotide polymorphism analysis) over a large temperature range, high sensitivity, and easy adaptation of conventional PCRs. Results: Using the LoopTag probe system we were able to detect all nine tested European species belonging to the Borrelia burgdorferi (sensu lato) complex and differentiated them from relapsing fever Borrelia species. As few as 10 copies of Borrelia in one PCR reaction were detectable. Conclusion: We established a novel multiplex probe real-time PCR system, designated LoopTag, that is simple, robust, and incorporates melting curve analysis for the detection and in the differentiation of European species belonging to the Borrelia burgdorferi s.l. complex.

Point-of-care molecular diagnosis of Mycoplasma pneumoniae including macrolide sensitivity using quenching probe polymerase chain reaction.

ObjectivesMacrolides are generally considered to be the drugs of choice for treatment of patients with Mycoplasma pneumoniae infection. However, macrolide-resistant M. pneumoniae has been emerging since about 2000. The Smart Gene® system (MIZUHO MEDY Co., Ltd., Tosu, Japan) is a novel fully automated system for detection of pathogens using the method of quantitative polymerase chain reaction (qPCR) with QProbe (QProbe PCR). The entire procedure is completed within 50 min and the size of the instrument is small (15 x 34 x 30 cm). The purpose of this study was to evaluate the usefulness of the Smart Gene® system for detection of M. pneumoniae and detection of a point mutation at domain V of the 23S rRNA gene of M. pneumoniae.MaterialsPharyngeal swab samples were collected from 154 patients who were suspected of having respiratory tract infections associated with M. pneumoniae.ResultsCompared with the results of qPCR, the sensitivity and specificity of the Smart Gene® system were 98.7% (78/79) and 100.0% (75/75), respectively. A point mutation at domain V of the 23S rRNA gene was detected from 7 (9.0%) of 78 M. pneumoniae-positive samples by the Smart Gene® system and these results were confirmed by direct sequencing. The minimum inhibitory concentrations of clarithromycin among the 5 isolates of M. pneumoniae with a point mutation at domain V of the 23S rRNA gene were >64 μg/ml and those among the 33 isolates without a mutation in the 23S rRNA gene were <0.0625 μg/ml.ConclusionThe Smart Gene® system is a rapid and accurate assay for detection of the existence of M. pneumoniae and a point mutation at domain V of the 23S rRNA gene of M. pneumoniae at the same time. The Smart Gene® system is suitable for point-of-care testing in both hospital and outpatient settings.

Virtual Fluorescence Color Channels by Selective Photobleaching in Digital PCR Applied to the Quantification of KRAS Point Mutations

Multiplexing of analyses is essential to reduce sample and reagent consumption in applications with large target panels. In applications such as cancer diagnostics, the required degree of multiplexing often exceeds the number of available fluorescence channels in polymerase chain reaction (PCR) devices. The combination of photobleaching-sensitive and photobleaching-resistant fluorophores of the same color can boost the degree of multiplexing by a factor of 2 per channel. The only additional hardware required to create virtual fluorescence color channels is a low-cost light-emitting diode (LED) setup for selective photobleaching. Here, we present an assay concept for fluorescence color multiplexing in up to 10 channels (five standard channels plus five virtual channels) using the mediator probe PCR with universal reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching characteristic of 21 URs, which cover the whole spectral range from blue to crimson. This comprehensive UR data set is employed to demonstrate the use of three virtual channels in addition to the three standard channels of a commercial dPCR device (blue, green, and red) targeting cancer-associated point mutations (KRAS G12D and G12V). Moreover, a LOD (limit of detection) analysis of this assay confirms the high sensitivity of the multiplexing method (KRAS G12D: 16 DNA copies/reaction in the standard red channel and KRAS G12V: nine DNA copies/reaction in the virtual red channel). Based on the presented data set, optimal fluorogenic reporter combinations can be easily selected for the application-specific creation of virtual channels, enabling a high degree of multiplexing at low optical and technical effort.

Whole-Gene Deletions of FZD4 Cause Familial Exudative Vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is an inherited disorder characterized by abnormalities in the retinal vasculature. The FZD4 gene is associated with FEVR, but the prevalence and impact of FZD4 copy number variation (CNV) on FEVR patients are unknown. The aim of this study was to better understand the genetic features and clinical manifestations of patients with FZD4 CNVs. A total of 651 FEVR families were recruited. Families negative for mutations in FEVR-associated genes were selected for CNV analysis using SeqCNV. Semiquantitative multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification were conducted to verify the CNVs. Four probands were found to carry whole-gene deletions of FZD4, accounting for 5% (4/80) of probands with FZD4 mutations and 0.6% (4/651) of all FEVR probands. The four probands exhibited similar phenotypes of unilateral retinal folds. FEVR in probands with CNVs was not more severe than in probands with FZD4 missense mutations (p = 1.000). Although this is the first report of FZD4 CNVs and the associated phenotypes, the interpretation of FZD4 CNVs should be emphasized when analyzing the next-generation sequencing data of FEVR patients because of their high prevalence.