Conventional Polymerase Chain Reaction Assays Research Articles

Detection of non-pathogenic and pathogenic populations of Vibrio parahaemolyticus in various samples by the conventional, quantitative and droplet digital PCRs.

In this study, three generations of polymerase chain reaction (PCR) assays: (i) conventional PCR, (ii) qPCR and (iii) droplet digital PCR (ddPCR), were systematically tested for their abilities to detect non-pathogenic and pathogenic populations of Vibrio parahaemolyticus. The limit of detection (LOD) for the ddPCR was 1.1pg/µL of purified DNA, followed by the qPCR (5.6pg/µL) and the conventional PCR (8.8pg/µL). Regarding the LOD for V. parahaemolyticus cells, the ddPCR assay was able to detect 29 cells, followed by the conventional PCR assay (58 cells) and the qPCR assay (115 cells). Regarding the sensitivities to detect this pathogen from PCR inhibition prone samples (naturally contaminated mussels), the ddPCR assay significantly outperformed the conventional PCR and qPCR. The ddPCR assay was able to consistently detect non-pathogenic and pathogenic populations of V. parahaemolyticus from naturally contaminated mussels, indicating its tolerance to various PCR inhibitors. This study also revealed the significant difference between conventional PCR and qPCR. The conventional PCR assay showed significantly greater sensitivity than that of the qPCR assay in detecting V. parahaemolyticus in crude samples, whereas the qPCR assay showed better sensitivity in detecting the presence of V. parahaemolyticus in purified DNA samples.

Evaluation of the diagnostic assays detecting red sea bream iridovirus infection at different severity levels

Red sea bream iridovirus (RSIV) is a highly contagious viral infection that affects various fish species and poses a significant threat to the global aquaculture industry. Thus, accurate and timely diagnosis is paramount for sustainable management of fish health. This study rigorously evaluated the diagnostic efficacy of various polymerase chain reaction (PCR) assays, focusing on those recommended by the World Organization for Animal Health (WOAH) and the assays newly proposed by WOAH's Aquatic Animals Health Standards Commission. Specifically, this study assessed conventional PCR, nested PCR, modified 1-F/1-R, and real-time PCR assays using a 95% limit of detection (LoD95%), as well as diagnostic sensitivity (DSe) and specificity (DSp) tests across different RSIV severity grades (G0−G4). In previous studies, the LoD95% for the 1-F/1-R and 4-F/4-R conventional assays were 225.81 and 328.7 copies/reaction, respectively. The modified 1-F/1-R exhibited a lower LoD95% of 51.32 copies/reaction. Notably, the nested PCR had an LoD95% of 11.23 copies/reaction, and the real-time PCR assay had an LoD95% of 12.02 copies/reaction. The DSe varied across RSIV severity grades, especially in the lower G0−G2 grades. The nested PCR and modified 1-F/1-R assays displayed the highest DSe, making them particularly useful for early-stage screening and detection of asymptomatic carriers. In addition, the PCR assays did not cross-react with any other aquatic pathogens except RSIV. Our findings significantly advanced the diagnostic capabilities of RSIVD by suggesting that nested PCR and modified 1-F/1-R assays are particularly promising for early detection. We propose their inclusion in future WOAH guidelines for a more comprehensive diagnostic framework.

Detection of feline immunodeficiency virus by neutral red-based loop-mediated isothermal amplification assay.

Feline immunodeficiency virus (FIV) is a retroviral pathogen globally responsible for immunodeficiency disease in cats. However, the current diagnosis based on antibody detection has limitations and can also produce false-positive results. This study aimed to develop a one-pot loop-mediated isothermal amplification (LAMP) process integrated with neutral red (NR-LAMP) assay for detection of FIV proviral DNA. We developed a one-pot, gag gene-based NR-LAMP for convenient, rapid, specific, and sensitive colorimetric inspection of FIV proviral DNA. The developed NR-LAMP was capable of amplifying at an optimum temperature of 65°C for 40 min. No cross-amplification was detected between FIV and other feline viruses tested, indicating the high specificity (98.44%) of the novel FIV-LAMP primer. Our NR-LAMP assay has a detection limit of 4.2 × 101 copies/μL. A total of 80 clinical samples with a background of FIV infection were collected and tested using the proposed method. The NR-LAMP assay showed a high sensitivity of 100% compared to conventional polymerase chain reaction assay. These results support the suitability of NR-LAMP as a potential future alternative clinical molecular approach for further use in the diagnosis of FIV-infected cats.

The Molecular Prevalence of Varicella-Zoster Virus and Human Herpesvirus Types 6 and 7 in Sperm Samples of Infertile Men

Background and Aims: Human herpes virus types 6 and 7 (HHV-6, HHV-7) can remain latent in the urogenital tract epithelial cells. Varicella-Zoster virus (VZV) is among the Alphaherpesviruses infecting most people during childhood. This virus can remain latent in the ganglionic neurons. The latent infection can reactivate through weakness, suppression of the immune system, and aging. Therefore, this study investigated the molecular prevalence of HHV-6, HHV-7, and Varicella-Zoster virus in sperm samples from infertile men.
 Materials and Methods: Sperm samples from 82 infertile patients were collected from infertility centers in Tehran. Chromatin health levels and sperm DNA examinations were carried out using sperm DNA fragmentation index kits. Viral nucleic acids were extracted from the samples. The presence of VZV, HHV-6, and HHV-7 genomes was examined through the conventional polymerase chain reaction assay.
 Results: The average age was 37.3 ± 6.1 years. The mean sperm motility percentage was 33.6 ± 2%. Infertile men's mean DNA fragmentation index was 27.2 ± 1.2. This study shows a 7.3% and 4.9% prevalence for HHV-6 and VZV, respectively, and the absence of HHV-7 in sperm specimens.
 Conclusion: Although the infection rates of these herpesviruses seem relatively low, more comprehensive and multi-center studies are required to obtain more accurate results.

First molecular detection of porcine circovirus type 4 (PCV4) in Malaysia.

Porcine circovirus type 4 (PCV4) is the newest member in the porcine circovirus family, first reported in 2020. To date, the presence of PCV4 has only been reported in China, South Korea and most recently in Thailand. Detection of PCV4 have been reported in various production stages of pigs from piglets, finishers to sows; associated with a myriad of clinical manifestations including porcine dermatitis and nephropathy syndrome (PDNS), postweaning multisystemic wasting syndrome (PMWS), respiratory, enteric and neurological diseases. While successful virus isolation and culture has yet to be reported, pathogenicity of PCV4 has been demonstrated through infectious clone studies. The objective of this study is to investigate the presence of PCV4 in Malaysian porcine population to update the epidemiology of porcine circoviruses in Malaysia. A total of 49 samples from commercial intensive pig farms, abattoir and wild boar population were subjected to conventional polymerase chain reaction assay to detect PCV4 capsid (cap) genome. Resulting cap nucleotide sequences were analyzed for maximum likelihood phylogeny relationship. Results revealed that PCV4 is present in Peninsular Malaysia at a molecular prevalence of 4.08% (2 / 49 samples). Both PCV4 positive samples originated from clinically healthy finishers. Malaysian PCV4 strains were classified as genotype PCV4b, and were found to be phylogenetically distinct from the China, South Korea and Thailand strains. With this latest update of the novel PCV4 in Malaysia, it is clear that more attention needs to be given to the investigation of novel porcine circoviruses (PCV) and management of PCV diseases.

Molecular characterization of Klebsiella pneumoniae associted with Thalassemia in Thi-Qar Governorate

Klebsiella pneumoniae is a gram-negative, aerobic, non-motile bacilli and is a common cause of a wide range of infections in humans, Klebsiella spp. as a major causative bacteria in Thalassemia patient. The present study aimed to shed light on the supposed role of bacterial infection in Thalassemia patients during the period of from August 2022 to December 2023 in Genetic Blood Diseases/Thi-Qar Governorate. Forty blood sample were collected from all patients and subjected to Conventional Polymerase Chain Reaction assay by using the Universal gene. Nine PCR product were selected and subjected to partial DNA sequencing for the 16S rRNA gene to follow up their possible relationship between them and what recorded globally in Gene bank. The results revealed that 9/40(25%) isolates were K. pneumoniae. PCR product of 16SrRNA registered in Gene bank under the official accession numbers of OQ928953.1,OQ929666.1,OQ928950.1,OQ929250.1,OQ927231.1,OQ928942.1,OQ927249.1,OQ927229.1 and OQ927253.1). The phylogenetic tree that was constructed by MEGA-10 software showed that there were different molecular relationships among the local K. pneumonia isolates with analogous ones around the world.

Development of a Multiplex PCR for Simultaneous Detection of Blueberry Red Ringspot Virus and Blueberry Scorch Virus Including an Internal Control

Blueberry red ringspot virus (BRRSV) and blueberry scorch virus (BlScV) are included in the quarantine virus list managed by the Korean Animal and Plant Quarantine Agency. A multiplex polymerase chain reaction (PCR) assay with an internal control was developed for the simultaneous detection of both viruses. The specific primers used here were designed based on the highly conserved regions of the genomic sequences of each virus, obtained from the National Center for Biotechnology Information nucleotide databases. The primers were designed to amplify a partial sequence within coat protein (CP) for detecting BRRSV and a partial sequence within the CP-16 kDa for detecting BlScV. 18S ribosomal RNA (rRNA) was used as internal control, and the primer set used in a previous study was modified in this study for detecting 18S rRNA. Each conventional PCR using the BRRSV, BlScV, and 18S rRNA primers exhibited a sensitivity of approximately 1 fg plasmid DNA. The multiplex PCR assay using the BRRSV, BlScV, and 18S rRNA primers was effective in simultaneously detecting the two viruses and 18S rRNA with a sensitivity of 1 fg plasmid DNA, similar to that of conventional PCR assays. The multiplex PCR assay developed in this study was performed using 14 blueberry cultivars grown in South Korea. BRRSV and BlScV were not detected, but 18S rRNA was all detected in all the plants tested. Therefore, our optimized multiplex PCR assay could simultaneously detect the two viruses and 18S rRNA in field samples collected from South Korea in a time-efficient manner. This approach could be valuable in crop protection and plant quarantine management.

Multicenter Testing of a Simple Molecular Diagnostic System for the Diagnosis of Mycobacterium Tuberculosis

Mycobacterium tuberculosis (MTB) is a communicable disease and still remains a threat to common health. Thus, early diagnosis and treatment are required to prevent the spread of infection. Despite the recent advances in molecular diagnostic systems, the commonly used MTB diagnostic tools are laboratory-based assays, such as mycobacterial culture, MTB PCR, and Xpert MTB/RIF. To address this limitation, point-of-care testing (POCT)-based molecular diagnostic technologies capable of sensitive and accurate detection even in environments with limited sources are needed. In this study, we propose simple tuberculosis (TB) molecular diagnostic assay by combining sample preparation and DNA-detection steps. The sample preparation is performed using a syringe filter with amine-functionalized diatomaceous earth and homobifunctional imidoester. Subsequently, the target DNA is detected by quantitative PCR (polymerase chain reaction). The results can be obtained within 2 h from samples with large volumes, without any additional instruments. The limit of detection of this system is 10 times higher than those of conventional PCR assays. We validated the clinical utility of the proposed method in 88 sputum samples obtained from four hospitals in the Republic of Korea. Overall, the sensitivity of this system was superior to those of other assays. Therefore, the proposed system can be useful for MTB diagnosis in limited-resource settings.

Molecular identification and phylogenetic confirmation of Sarcocystis species in slaughtered camels in Al-Najif province, Iraq.

Sarcocystis is an intracellular parasite of particular importance as it infects many domestic animals as camels that play the role of intermediate host for the parasite. This study aimed to identify Sarcocystis species in camels by molecular assay with confirmation of local isolates by phylogenetic analysis. A total of 200 slaughtered camels (Camelus dromedarius) that were slaughtered in Al-Najaf province (Iraq) abattoirs from October (2021) to July (2022) were subjected to collect the fresh tissues from four organs (esophagus, diaphragm, skeletal muscle, and heart), to be tested later by the conventional polymerase chain reaction (PCR). Then, a total of 20 positive genomic DNA samples were sequenced, named, got specific access numbers (OP785703.1 to OP785722.1), and compared with the NCBI-GenBank isolates. Targeting Cox1 gene, 80% of collected tissues were found positive by the conventional PCR assay. Phylogenetic tree analysis revealed that the local Sarcocystis isolates were identical to Indian S. cameli isolates at 99.70%-99.90%. Significantly, an increase in Sarcocystis infection was seen in the esophagus compared to the diaphragm, skeletal muscle, and heart; older (>4 years) than younger (≤4 years) camels, and in females more than males. To the best of our knowledge, this represents the first molecular study in Iraq that identifies Sarcocystis cameli in camels. However, additional epidemiological and molecular studies in camel populations as well as in other domestic and wild animals appeared to be necessary.

Solvent-sensitive nanoparticle-enhanced PCR assay for the detection of enterotoxigenic Escherichia coli

Stimulus-responsive nanoparticles are among the most utilized nanoscale materials in biomedical applications. As these nanoparticles exhibit a manipulable response to a particular stimulus, such as pH, heat, and organic solvent, they are potential signalling units in diagnostic assays. This study aims to enhance the limit of detection and reduce the turnaround time of magnetic nanoparticle polymerase chain reaction (PCR) enzyme-linked gene assay (MELGA), an advanced PCR-based technique termed the solvent-sensitive nanoparticle (SSNP)-enhanced PCR assay. This technique was proposed to detect pathogenic enterotoxigenic Escherichia coli (ETEC) through applying stimulus-responsive nanoparticles. The SSNPs were elaborated with three main components, including mesoporous silica nanoparticles as a structural unit, organic dye (Nile red) as a payload, and the corresponding organic solvent-sensitive polymer shell as “gatekeeper” (poly(maleic anhydride-alt-methyl vinyl ether, PMAMVE). A suitable organic solvent capable of inducing polymer swelling and dye dissolution was investigated by considering a solubility parameter. Using ethanol, the encapsulated Nile red can diffuse out of the SSNPs faster than other solvents and reach a constant concentration within 15 min. For the PCR inhibition study, various SSNPs concentrations (10–30 μg/reaction) were mixed with the ETEC gene and PCR reagent. The results showed that the particles in this concentration range did not inhibit PCR. By comparing the efficacy of conventional PCR, MELGA, and SSNP-enhanced PCR assay, the proposed technique showed a better detection limit than that of PCR, whereas that of MELGA was the lowest. Moreover, compared to MELGA or conventional PCR, this technique provided remarkably faster results in the postamplification process.

Comparison of ELISA and PCR Assays for Detection of Pork Adulteration in Halal-Labelled Beef Products

Background: Food adulteration with pork in processed beef products is one of the most serious issues in a food sector in a Muslim-majority country since it is related to religious food ethics regarding the halal products. The goal of this research is to test the suitability of ingredients in beef floss and its Halal by knowing the presence of porkDNA and protein in those products.
 Methods: Meat products were prepared from two famous marketplaces in Indonesia labeled contain beef meat. In this study, a qualitative Enzyme-Linked Immunosorbent Assay (ELISA) test was compared to a conventional Polymerase Chain Reaction (PCR) assay to determine pork adulteration in beef floss.
 Results: The results of the ELISA test showed that two products labeling Halal and containing beef ingredients were positive for pork. Those two samples continued testing using conventional PCR assay. The result of the conventional PCR assay was negative for those two samples.
 Conclusion: It may be helpful to utilize both traditional PCR and ELISA for species detection due to the possibly inhibiting compounds contained in some processed meat products. The results of this research suggest that ELISA is better than conventional PCR method for product samples that have received an intensive heating process.

Development and Evaluation of NanoPCR for the Detection of Goose Parvovirus.

Simple SummaryGosling plague (GP), an acute, virulent infectious disease caused by goose parvovirus (GPV), is a serious problem for livestock and poultry breeding. At present, there is no effective treatment available. The disease is vertically transmitted in geese, and some infected pregnant females are often recessive carriers of the virus, making it very difficult for farmers to detect GPV in the clinical setting. Although there are many clinical testing methods for GPV, some of them still suffered from shortcomings such as being time-consuming and labor-intensive. In this study, gold nanoparticles were put into a conventional PCR reaction system, and the first nanoPCR reaction was successfully established to detect infected GPV in the clinic, thus providing a practical method for the detection of GPV clinical infection.Gosling plague (GP) is an acute and hemorrhagic infectious disease caused by goose parvovirus (GPV). The goose industry suffers significant economic losses as a result of GP, which is found to be widespread worldwide, with high rates of morbidity and mortality. Our group developed a novel technique for detecting GPV nanoparticle-assisted polymerase chain reaction (nanoPCR) and the characterization of its specificity and sensitivity. It was developed by using the traditional polymerase chain reaction (PCR) and nanoparticles. The findings of this study revealed that GPV nanoPCR products were 389 bp in length, and the lower limit of the nanoPCR assay was 4.68 × 102 copies/μL, whereas that of the conventional PCR assay was 4.68 × 104 copies/μL. A total of 230 geese suspected of GPV were detected using nanoPCR, with a positive rate of 83.0% and a specificity of 73%, respectively. Overall, we present a hitherto undocumented method for identifying GPV by using nanoPCR to aid in the evaluation of subclinical illness.

Diagnostic Performance and Clinical Utility of Conventional PCR Assay in Early Diagnosis of COVID-19 Associated Rhino-Orbito-Cerebral Mucormycosis.

Early diagnosis and treatment of rhino-orbital-cerebral mucormycosis (ROCM) are crucial. Potassium hydroxide with Calcofluorwhite (KOH + CFW) smears can demonstrate the fungal hyphae, but mixed infections caused by both mucorales and non-mucorales pose a diagnostic challenge. Polymerase chain reaction (PCR) can detect mixed infections and differentiate mucorales from non-mucorales. This study aimed to evaluate the utility of a single reaction PCR in the diagnosis of ROCM and the efficacy of nasal biopsy and endonasal swab in the detection of fungus. Sixty-six clinical samples were collected from 33 patients and were subjected to KOH + CFW smear, culture and PCR. PCR was performed using pan-fungal primers targeting the 28S large subunit rRNA gene, and the amplified products were further sequenced to identify the fungi. KOH + CFW smear, culture and PCR detected mucorales in 54.6%, 27.3% and 63.6% patients, respectively. PCR detected mixed infection in 51.5% patients compared to 9.1% by KOH + CFW smear. PCR detected fungus in 90% of nasal biopsies and 77.8% of endonasal swabs. Rhizopus spp. was the most common fungi identified in 43.2% of PCR-positive samples. PCR is effective in detecting mixed infection and in the diagnosis of ROCM. Nasal biopsies had better fungal detection rates than endonasal swabs.

Phenotypic and molecular characterization of extended-spectrum β-lactamase/AmpC- and carbapenemase-producing Klebsiella pneumoniae in Iran.

The objective of the current study is to evaluate the phenotypic and molecular characterization of ESBL/AmpC- and carbapenemase-producing K. pneumoniae isolates in Iran. From October 2018 until the end of April 2020, different clinical samples were collected and K. pneumoniae isolates were identified using conventional biochemical tests and PCR assay. Antibiotic susceptibility pattern was determined using the Kirby-Bauer disk diffusion method. Modified Hedge Test (MHT) was applied to the identification of carbapenemase-producing K. pneumoniae. ESBL and AmpC-producing K. pneumoniae were detected using Double Disc Test (DDT) and Disc Potentiation Test (DPT), respectively. The presence of carbapenemase, ESBL, and AmpC encoding genes was screened by Polymerase Chain Reaction (PCR) assay. A total of 100 K. pneumoniae isolates were collected. K. pneumoniae isolates had the highest resistance rate to cefazolin (66%) and cefotaxime (66%). Meropenem and amikacin with sensitivity rates of 76% and 69% were the most effective antimicrobial agents on K. pneumoniae isolates. It was found that 12 (12%), 27 (27%), and 9 (9%) K. pneumoniae isolates were positive in MHT, DDT, and DPT tests, respectively. Among the carbapenemase-encoding genes, blaOXA-48 (24%) and blaIMP (13%) genes had the highest frequency, while blaKPC and blaGIM genes were not detected among K. pneumoniae isolates. blaTEM (48%) and blaCMY (8%) genes had the highest frequency among ESBL and AmpC β-lactamase-encoding genes, respectively. It is vital to adopt effective control strategies for K. pneumoniae infections and ensure rapid identification of antibiotic resistance profile.

MOLECULAR PCR ASSAYS FOR DETECTION OF Ganoderma PATHOGENIC TO OIL PALM IN MALAYSIA

Ganoderma boninense is a fungal pathogen that causes basal stem rot (BSR) disease in oil palm. Being a serious disease problem to the oil palm industry, monitoring and detecting the pathogen is of the utmost importance to reduce disease spread and facilitate effective management strategies. Because traditional culture-based assay is time-consuming, labour-intensive and required special skills in mycology, plant pathologists are turning to more accurate, sensitive and fast methods such as molecular techniques. In this study, polymerase chain reaction (PCR) assays were developed to detect pathogenic Ganoderma species causing BSR disease in oil palm using a primer designed based on the ribosomal DNA internal transcribed spacer (ITS) region. The effectiveness of conventional and real-time PCR assays was analysed compared to the traditional isolation-based assay. For artificially inoculated oil palm plantlets, consistent detection of G. boninense was observed. Real-time PCR assay has shown to be more sensitive and rapid in detecting G. boninense in field samples and could potentially serve as a validation tool to other detection techniques for implementation of effective disease control measures.

Molecular detection of H1N1 virus by conventional reverse transcription PCR coupled with nested PCR

Influenza A pdm09 virus has been spreading worldwide and creates a serious public health threat. Among different subtypes/lineages of swine influenza A viruses, the H1N1 subtype is more prevalent in all-over world infections followed by H3N2 which is present in the northern hemisphere. Influenza A pdm09 is dominant for centuries but the last pandemic was in 2009 and is responsible for 151,700–575,400 deaths within one year worldwide (Centres for Disease Control and Prevention). Rapid diagnosis for H1N1 is essential which must be highly sensitive and specific and can differentiate between influenza A subtypes and other respiratory pathogens. In this study, a conventional polymerase chain reaction assay and nested polymerase chain reaction assay were developed which can detect H1N1 pdm09. One outer set of primer is designed and one inner set of primer is designed specifically for hemagglutinin gene (H1). Specificity was checked using different pathogenic genomes for amplification of the H3N2 subtype, Neisseria meningitides, Salmonella typhi, etc. The sensitivity of the nested polymerase chain reaction is 0.001ng/6 ​μL of PCR amplified product of outer primer product of HA1 gene as compared to 50 ​ng/μL of two-step conventional polymerase chain reaction. The nested PCR and conventional PCR are performed on 80 samples which are consistent with the Real-Time PCR results. Real-Time is a standard test for H1N1 detection. But it is very expensive but current method is less expensive and provides comparable results with Real-Time PCR.

PCR-based Evaluation of Human Papillomavirus Genotypes in Oral Lichen Planus.

Objective:The objective of the study was to use polymerase chain reaction (PCR) to examine and compare the genotype distribution of human papillomavirus (HPV) in oral lichen planus (OLP).Materials and Methods:Deoxyribonucleic acid (DNA) was extracted from 20 OLP biopsy specimens. Conventional PCR assay employing consensus HPV primers was used to identify HPV DNA. Positive PCR samples were then subjected to PCR assay with HPV type-specific primers.Results:Out of the total 20 OLP specimens evaluated, eight samples (40%) were positive for HPV. Females had a 41.7% higher HPV-positive rate than males. The most common type in the HPV type-specific PCR assay was HPV-18 (75%), which is a high-risk type of HPV linked to malignant diseases. The erosive kind of OLP had the greatest percentage of HPV positives (50%).Conclusion:The present study confirms the detection of HPV in OLP lesions, as determined by PCR-coupled HPV gene sequencing, as well as its likely mechanism of malignant transformation.

Diagnosis of Lyme Borreliosis With a Novel, Seminested Real-Time Polymerase Chain Reaction Targeting the 5S-23S Intergenic Spacer Region: Clinical Features, Histopathology, and Immunophenotype in 44 Patients.

Lyme borreliosis (LB) is the most common tick-borne infection in Europe and North America. Polymerase chain reaction (PCR) is an important tool to confirm the diagnosis, but not always successful, especially when organisms are sparse. We developed a novel, seminested real-time PCR assay [target: 5S-23S intergenic spacer region (IGS)] and compared it with 3 well-established conventional PCR assays (IGS/OspA/real-time IGS) on 596 formalin-fixed, paraffin-embedded routine skin biopsies. The seminested real-time assay identified 46 cases of borreliosis while 25, 27, and 38 were identified by the 3 other assays, respectively (P 0.01, P 0.02, and P 0.42; significance P < 0.05). Clinicopathologic and immunophenotypic analysis of PCR-positive cases revealed 38 erythema migrans (EM), 6 Borrelia lymphocytomas, and 2 acrodermatitis chronica atrophicans (ACA). In the 44 PCR-confirmed cases, plasma cells were present in only a third of EM cases. By contrast, CD123-positive plasmacytoid dendritic cells were common (74%) and therefore are unlikely to be helpful in the differential diagnosis between EM and tumid lupus erythematosus. A loss of CD34 in a third of all LB specimens limits its diagnostic value in the differential diagnosis with morphea. Interstitial macrophages were common in cutaneous LB (42/43) forming interstitial granulomas in a third of all cases, and 3/38 EM, 3/6 Borrelia lymphocytomas, and 1/2 ACA were only identified by the new seminested real-time assay, suggesting that it is especially helpful in confirming the diagnosis of Borrelia lymphocytoma.

Evaluation of a novel TaqMan probe-based real-time polymerase chain reaction (PCR) assay for detection and quantitation of red sea bream iridovirus

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD95%) was determined to be 5.3 viral genome copies/μL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

Detection of Sri Lankan cassava mosaic virus by loop-mediated isothermal amplification using dried reagents

Recently, the widespread occurrence of Sri Lankan cassava mosaic virus (SLCMV), genus Begomovirus, family Geminiviridae, which causes a mosaic disease in cassava (Manihot esculenta Crantz) in South-East Asia have, become a serious economic issue. Since cassava is propagated through vegetative cuttings, a rapid virus diagnostic method is crucial for generating virus-free planting materials. In this study, a loop-mediated isothermal amplification (LAMP) assay using six primers was developed and validated for the rapid detection of SLCMV in cassava leaves. This SLCMV assay had a detection sensitivity that was up to 10,000 times higher than that of the conventional polymerase chain reaction assay and can detect the virus from symptomless stem cutting, which is a potential long-distance spreader of the virus. Furthermore, a practical LAMP protocol using stable dried reagents from a commercial kit was established so that the assay could be performed in the field by incubating the reactions in water at 60–65 °C instead of using a thermal cycler. The primer sequences and the LAMP protocol described here should be useful for the rapid and sensitive on-site detection of SLCMV.