Pathogen-specific Primers Research Articles

Metagenomic versus targeted next-generation sequencing for detection of microorganisms in bronchoalveolar lavage fluid among renal transplantation recipients.

Metagenomic next-generation sequencing (mNGS), which provides untargeted and unbiased pathogens detection, has been extensively applied to improve diagnosis of pulmonary infection. This study aimed to compare the clinical performance between mNGS and targeted NGS (tNGS) for microbial detection and identification in bronchoalveolar lavage fluid (BALF) from kidney transplantation recipients (KTRs). BALF samples with microbiological results from mNGS and conventional microbiological test (CMT) were included. For tNGS, samples were extracted, amplified by polymerase chain reaction with pathogen-specific primers, and sequenced on an Illumina Nextseq. A total of 99 BALF from 99 KTRs, among which 93 were diagnosed as pulmonary infection, were analyzed. Compared with CMT, both mNGS and tNGS showed higher positive rate and sensitivity (p<0.001) for overall, bacterial and fungal detection. Although the positive rate for mNGS and tNGS was comparable, mNGS significantly outperformed tNGS in sensitivity (100% vs. 93.55%, p<0.05), particularly for bacteria and virus (p<0.001). Moreover, the true positive rate for detected microbes of mNGS was superior over that of tNGS (73.97% vs. 63.15%, p<0.05), and the difference was also significant when specific for bacteria (94.59% vs. 64.81%, p<0.001) and fungi (93.85% vs. 72.58%, p<0.01). Additionally, we found that, unlike most microbes such as SARS-CoV-2, Aspergillus, and EBV, which were predominantly detected from recipients who underwent surgery over 3 years, Torque teno virus (TTV) were principally detected from recipients within 1-year post-transplant, and as post-transplantation time increased, the percentage of TTV positivity declined. Although tNGS was inferior to mNGS owing to lower sensitivity and true positive rate in identifying respiratory pathogens among KTRs, both considerably outperformed CMT.

Molecular rapid diagnostic testing for bloodstream infections: Nanopore targeted sequencing with pathogen-specific primers

Nanopore sequencing, known for real-time analysis, shows promise for rapid clinical infection diagnosis but lacks effective assays for bloodstream infections (BSIs). We prospectively assessed the performance of a novel nanopore targeted sequencing (NTS) assay in identifying pathogens and predicting antibiotic resistance in BSIs, analyzing 387 blood samples from December 2021 to April 2023. The positivity rate for NTS (69.5%, 269/387) nearly matches that of metagenomic next-generation sequencing (mNGS) (74.7%, 289/387; p=0.128) and surpasses the positivity rate of conventional blood culture (BC) (33.9%, 131/387; p<0.01). Frequent pathogens detected by NTS included Klebsiella pneumoniae (n=54), Pseudomonas aeruginosa (n=36), Escherichia coli (n=36), Enterococcus faecium(n=30), Acinetobacter baumannii(n=26), Staphylococcus aureus(n=23), and Human cytomegalovirus (n=37). Against a composite BSI diagnostic standard, NTS demonstrated a sensitivity and specificity of 84.0% (95% CI 79.5%-87.7%) and 90.1% (95% CI 81.7%-88.5%), respectively. The concordance between NTS and mNGS results (the percentage of total cases where both either detected BSI-related pathogens or were both negative) was 90.2% (359/387), whereas the consistency between NTS and BC was only 60.2% (233/387). In 80.6% (50/62) of the samples with identical pathogens identified by both NTS tests and BCs, the genotypic resistance identified by NTS correlated with culture-confirmed phenotypic resistance. Using NTS, 95% of samples can be tested and analyzed in approximately 7h, allowing for early patient diagnosis. NTS is rapid, sensitive, and efficient for detecting BSIs and drug-resistant genes, making it a potential preferred diagnostic tool for early infection identification in critically ill patients.

Molecular screening of chewing sticks and sponges found on the ghanaian local market for diarrhoea-causing microbes- a pilot study

Chewing sticks and sponges are used for oral hygiene in Ghana and other African countries. In addition to their affordability, they have additional advantages of anti-microbial and an­ti-plague properties. They are usually sold on the open market under low hygienic conditions, exposing them to environmental pathogens. Since they are mostly not sterilized before use, it is important to screen for the presence of pathogens of public health importance on randomly selected samples. This preliminary study used molecular assays to screen for Rotavirus A, &lt;i&gt;Sal­monella&lt;/i&gt; &lt;i&gt;typhi&lt;/i&gt;, &lt;i&gt;Vibrio&lt;/i&gt; &lt;i&gt;cholera&lt;/i&gt; and &lt;i&gt;Escherichia&lt;/i&gt; &lt;i&gt;coli&lt;/i&gt; on 10 chewing stick and sponge samples purchased randomly from the Agbogbloshie market in Accra. The samples were incubated in sterile distilled water overnight at room temperature to dislodge pathogens. Dislodged patho­gens were captured using the Nanotrap Microbiome A kit. Total nucleic acids were extracted from concentrates using the RADI prep DNA/RNA kit. All PCR assays were performed using 2X SYBR Green Mix and pathogen specific primers. Out of the four pathogens screened, only &lt;i&gt;E.&lt;/i&gt; &lt;i&gt;coli&lt;/i&gt; was detected (40% and 60% of chewing sponge and stick samples respectively). Despite the advantages of chewing sticks and sponges, the detection of &lt;i&gt;E.&lt;/i&gt; &lt;i&gt;coli&lt;/i&gt; on samples is a cause for concern since they indicate faecal contamination and can cause diarrhoeal diseases. It is recommended that chewing sticks and sponges should be washed clean before used for oral health. An alternative is to train local producers and retailers on improved hygienic pack­aging and handling of these essential cleaning agents.&#x0D;

AI-driven colorimetric nucleic acid test for tilapia lake virus: A large-scale, point-of-care diagnostic model for future emerging diseases

It is indisputable that the world is currently facing several health challenges, which include outbreaks of diseases. One way to limit the spread of such diseases is to have simple yet efficient bioassays that work well in large-scale operation. To address this issue, a new artificial intelligence (AI) based colorimetric DNA/RNA detection platform was developed using tilapia lake virus as a model. The assay uses a newly formulated indicator dye and colorimetric nucleic acid amplification done in a thermos or a simple heating block to produce visible results that can be observed by the naked eye. To ensure high-throughput screening, an AI-based analysis web application was developed that can be used on computers, tablets, and smartphones to accurately and quickly determine the colorimetric results. With a genetic material extraction procedure that takes only 5 min and requires no equipment, our assay can be completed within an hour from sampling to readout, with 96% accuracy and a sensitivity approaching a single copy of the target virus. The entire assay incubation can be done in a simple thermos, as a substitute for thermal cyclers, rendering its potential use in remote areas where access to electricity is restricted. To demonstrate the versatility of our platform, we modified our testing solution by changing pathogen-specific primers and tested it on various target pathogens that infect humans, animals, and plants. The results were accurate, indicating that the platform has the potential to be used for point-of-care disease diagnosis in the future.

Development of simple and quick DNA release protocol for conventional PCR based detection of Xanthomonas axonopodis pv. punicae causing bacterial blight in pomegranate

An economical, simple, rapid, and culture independent method was developed for routine analyses and detection of Xanthomonas axonopodis pv. punicae (Xap) that causes bacterial blight in pomegranate. Five DNA release buffers (B1-B5) were optimized for extracting bacterial genomic DNA (gDNA) directly from (a)symptomatic pomegranate leaves followed by conventional polymerase chain reaction (PCR)-based detection of Xap. B1, B3, and B4 were found suitable to release gDNA, which was subjected to PCR using universal primers for 16S rRNA and rpsL genes, and pathogen specific xopQ primers. DNA released from B1 and B4 successfully produced amplicons of expected sizes. Additional analyses found that DNA released using B4 buffer was stable up to 45 days at −20 °C/−80 °C and 35 days at 4 °C and 8-800 pg DNA could be detected by the PCR-based assay. B4 was further validated for versatility by extracting DNA of Xanthomonas spp. causing citrus canker and sorghum shoot stripe disease.

Differential diagnosis of DNA viruses related to reproductive disorder on sows by multiplex-PCR technique

The newly emerged diseases caused by ASFV and PCV3 and their confirmed prevelance in Vietnam whereas most of available common commercial methods such as ELISA or realtime PCR designed for detecting single pathogen per reaction, highlighted a necessity for another diagnostic method to simultaneously detect and differentiate DNA viruses that are related to reproductive failures in sow herds including PCV2, PCV3, PPV, ASFV. In this communication, a diagnostic multiplex-PCR (mPCR) was established with pathogen-specific primers selected from previous studies and another set of primers designed for COX1 gene serving as an internal amplification control (IAC). The predicted products of PCV2, PCV3, PPV, ASFV and IAC were 702 bp, 223 bp, 380 bp, 278 bp and 463 bp, respectively. After optimization, the mPCR functioned specifically at 62°C. Results revealed the consistent detection limit at 100 copies/gene/reaction. In application, 185 serum samples from sows were used to examine the presence of the related pathogens. mPCR results showed that the mono-infection rate of PCV2, PCV3, PPV, and ASFV was 0% (0/185), 40% (74/185), 28.1% (52/185), and 48.1% (89/185), respectively. Regarding coinfection rate, the data indicated that coinfections of 2, 3 and 4 pathogens were 20%, 8.1% and 0% accordingly. In conclusion, the mPCR assay was successfully established and ready to serve for diagnosis of PCV2, PCV3, PPV and ASFV infection in reality with high specificity and sensitivity. It is a good contribution to a better understanding of the epidemiology of these diseases in swine.

Monitoring of ticks and their pathogens from companion animals obtained by the “tekenscanner” application in The Netherlands

Ticks are vectors for many pathogens of veterinary and medical interest. In order to monitor ticks and tick-borne pathogens, the “Tekenscanner” (Dutch for Tick scanner), a citizen science project, was launched in The Netherlands. It is a smartphone application for pet-owners to get ticks from their dog or cat, identified and checked for pathogens for free. At the same time, information about the pet and the geographic location of tick infestation becomes available for research. The application was launched in 2018, and the results of the first 6 months after launch of the app were reported. Ticks were identified based on morphology, and DNA was extracted and amplified by a panel of tick-borne pathogen-specific primers. Next, the amplicons were subjected to reverse line blot with specific probes for important pathogens to determine their presence or absence. The present paper describes the results of 2019 and 2020. There were 2260 ticks collected from 871 dogs and 255 cats (26 ticks were from an unknown host) and all pet owners were informed about the results. Four species of ticks were collected: Ixodes ricinus (90.0%), Ixodes hexagonus (7.3%), Dermacentor reticulatus (2.8%) and Rhipicephalus sanguineus (0.1%). Ixodes ricinus was the tick with the most divergent pathogens: Anaplasma sp. (1.3%), Babesia sp. (0.8%), Borrelia spp. (4.8%), Neoehrlichia sp. (3.7%) and Rickettsia helvetica (12.6%). In I. hexagonus, R. helvetica (1.8%) and Babesia sp. (0.6%) were detected and Rickettsia raoultii in D. reticulatus (16.2%). One of the two nymphs of R. sanguineus was co-infected with Borrelia and R. helvetica and the other one was uninfected. The high numbers of different pathogens found in this study suggest that companion animals, by definition synanthropic animals, and their ticks can serve as sentinels for emerging tick-borne pathogens.

TASPERT: Target-Specific Reverse Transcript Pools to Improve HTS Plant Virus Diagnostics.

High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique’s success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.

In Silico Study of Developing a Method for Detecting Pathogenic Bacteria in Refillable Drinking Water Samples

One of the parameters of quality of drinking water that is suitable for consumption is not contaminated by Escherichia coli, Salmonella and Shigella sp. Beside microbiological tests, water quality testing can be carried out molecularly only in few hours, using PCR (Polymerase Chain Reactions) technique. Although PCR is a basic molecular technique, various detection methods can be developed from it. The key to success in making a PCR-based method is inseparable from bioinformatics studies when designing primers that are specific to pathogen target DNA. The purpose of this research was to design pathogen-specific primers and do in silico study of PCR using bioinformatics software to get better planning in developing detection method. DNA sequence templates of E. coli, Salmonella and Shigella were downloaded from NCBI and multiple-aligned using Geneious Prime bioinformatic software. Primers were designed according to conserved region of these pathogens. The primers specificity was checked using Primer BLAST tools in NCBI. The result of this study was a pair of primers that amplify 825 bp fragment of 16S rRNA sequence specific to E. coli, Salmonella and Shigella.

Whole genome analysis of the koa wilt pathogen (Fusarium oxysporum f. sp. koae) and the development of molecular tools for early detection and monitoring

BackgroundDevelopment and application of DNA-based methods to distinguish highly virulent isolates of Fusarium oxysporum f. sp. koae [Fo koae; cause of koa wilt disease on Acacia koa (koa)] will help disease management through early detection, enhanced monitoring, and improved disease resistance-breeding programs.ResultsThis study presents whole genome analyses of one highly virulent Fo koae isolate and one non-pathogenic F. oxysporum (Fo) isolate. These analyses allowed for the identification of putative lineage-specific DNA and predicted genes necessary for disease development on koa. Using putative chromosomes and predicted gene comparisons, Fo koae-exclusive, virulence genes were identified. The putative lineage-specific DNA included identified genes encoding products secreted in xylem (e. g., SIX1 and SIX6) that may be necessary for disease development on koa. Unique genes from Fo koae were used to develop pathogen-specific PCR primers. These diagnostic primers allowed target amplification in the characterized highly virulent Fo koae isolates but did not allow product amplification in low-virulence or non-pathogenic isolates of Fo. Thus, primers developed in this study will be useful for early detection and monitoring of highly virulent strains of Fo koae. Isolate verification is also important for disease resistance-breeding programs that require a diverse set of highly virulent Fo koae isolates for their disease-screening assays to develop disease-resistant koa.ConclusionsThese results provide the framework for understanding the pathogen genes necessary for koa wilt disease and the genetic variation of Fo koae populations across the Hawaiian Islands.

Cross-kingdom analysis of nymphal-stage Ixodes scapularis microbial communities in relation to Borrelia burgdorferi infection and load.

The tick microbiota may influence the colonization of Ixodes scapularis by Borrelia burgdorferi, the Lyme disease bacterium. Using conserved and pathogen-specific primers we performed a cross-kingdom analysis of bacterial, fungal, protistan and archaeal communities of I. scapularis nymphs (N = 105) collected from southern Vermont, USA. The bacterial community was dominated by a Rickettsia and several environmental taxa commonly reported in I. scapularis, as well as the human pathogens B. burgdorferi and Anaplasma phagocytophilum, agent of human granulocytic anaplasmosis. With the fungal primer set we detected primarily plant- and litter-associated taxa and >18% of sequences were Malassezia, a fungal genus associated with mammalian skin. Two 18S rRNA gene primer sets, intended to target protistan communities, returned mostly Ixodes DNA as well as the wildlife pathogen Babesia odocoilei (7% of samples), a Gregarines species (14%) and a Spirurida nematode (18%). Data from pathogen-specific and conserved primers were consistent in terms of prevalence and identification. We measured B. burgdorferi presence/absence and load and found that bacterial beta diversity varied based on B. burgdorferi presence/absence. Load was weakly associated with bacterial community composition. We identified taxa associated with B. burgdorferi infection that should be evaluated for their role in vector colonization by pathogens.

Passive Tick Surveillance: Exploring Spatiotemporal Associations of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), Babesia microti (Piroplasmida: Babesiidae), and Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) Infection in Ixodes scapularis (Acari: Ixodidae).

Ixodes scapularis transmits a group of pathogens, including Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum, the causative agents for Lyme disease, babesiosis, and anaplasmosis, respectively. I. scapularis ticks submitted by state residents to the Connecticut Agricultural Experiment Station-Tick Testing Laboratory between 2015 and 2018 were screened using standard PCR and pathogen-specific primers. Infection and coinfection prevalence in I. scapularis was estimated to assess differences in infection status by life stage (nymph or adult female), county, and year, as well as whether infection with B. burgdorferi changes the likelihood of infection with either B. microti or A. phagocytophilum. Of the 11,254 I. scapularis acquired in Connecticut, 40.7% tested positive for at least one pathogen and the remaining 59.3% were negative. Most I. scapularis ticks tested positive for a single pathogen (33.6%), and only 7.2% were infected with more than one pathogen, of which 93.2% were identified with dual infection and 6.8% tested positive for all three pathogens. Adults were more likely than nymphs to be infected or coinfected with these pathogens. Furthermore, we found that ticks were 74% more likely to be infected with B. microti and 98% more likely to be infected with A. phagocytophilum if infected with B. burgdorferi compared with those not infected. We did not find spatial differences in infection or coinfection prevalence, but between 2015 and 2018, the likelihood that a tick was coinfected increased with time. These results from Connecticut, an endemic state for Lyme disease with long-established populations of I. scapularis, suggest that the increased likelihood of coinfection prevalence over time may have significant implications for clinical diagnosis, course, severity, and treatment of human disease cases.

Development and application of two multiplex real-time PCR assays for detection and speciation of bacterial pathogens in the koala.

Infectious diseases have contributed to the decline in the health of koala ( Phascolarctos cinereus) populations in the wild in some regions of Australia. Herein we report the development and validation of 2 multiplex real-time PCR (rtPCR) panels for the simultaneous detection of Mycoplasma spp., Ureaplasma spp., Bordetella bronchiseptica, and Chlamydia, including speciation and quantification of Chlamydia, in ocular, reproductive, and nasal swab samples in addition to semen and male urogenital and reproductive tissues, from koalas. Each rtPCR panel was developed for use as a single-tube reaction using pathogen-specific primers and fluorescently labeled probe sets. DNA extracted from reference strains and isolates was used for validation of sequence gene targets for the multiplex rtPCR panels. Each panel was shown to be sensitive and specific in detecting and differentiating the bacterial pathogens. The multiplex rtPCR panels were used to screen clinical samples from free-ranging and hospitalized koalas for multiple pathogens simultaneously. The multiplex rtPCR will improve turnaround time compared to individual-pathogen rtPCR methods used, to date, for confirmation of diagnosis and will provide the wildlife clinician with the ability to make treatment decisions more rapidly.

Disclosing respiratory co-infections: a broad-range panel assay for avian respiratory pathogens on a nanofluidic PCR platform

ABSTRACT Respiratory syndromes (RS) are among the most significant pathological conditions in edible birds and are caused by complex coactions of pathogens and environmental factors. In poultry, low pathogenic avian influenza A viruses, metapneumoviruses, infectious bronchitis virus, infectious laryngotracheitis virus, Mycoplasma spp. Escherichia coli and/or Ornithobacterium rhinotracheale in turkeys are considered as key co-infectious agents of RS. Aspergillus sp., Pasteurella multocida, Avibacterium paragallinarum or Chlamydia psittaci may also be involved in respiratory outbreaks. An innovative quantitative PCR method, based on a nanofluidic technology, has the ability to screen up to 96 samples with 96 pathogen-specific PCR primers, at the same time, in one run of real-time quantitative PCR. This platform was used for the screening of avian respiratory pathogens: 15 respiratory agents, including viruses, bacteria and fungi potentially associated with respiratory infections of poultry, were targeted. Primers were designed and validated for SYBR green real-time quantitative PCR and subsequently validated on the Biomark high throughput PCR nanofluidic platform (Fluidigm©, San Francisco, CA, USA). As a clinical assessment, tracheal swabs were sampled from turkeys showing RS and submitted to this panel assay. Beside systematic detection of E. coli, avian metapneumovirus, Mycoplasma gallisepticum and Mycoplasma synoviae were frequently detected, with distinctive co-infection patterns between French and Moroccan flocks. This proof-of-concept study illustrates the potential of such panel assays for unveiling respiratory co-infection profiles in poultry.

Molecular diagnosis of central nervous system opportunistic infections and mortality in HIV-infected adults in Central China

BackgroundCSF PCR is the standard diagnostic technique used in resource-rich settings to detect pathogens of the CNS infection. However, it is not currently used for routine CSF testing in China. Knowledge of CNS opportunistic infections among people living with HIV in China is limited.MethodsIntensive cerebrospiral fluid (CSF) testing was performed to evaluate for bacterial, viral and fungal etiologies. Pathogen-specific primers were used to detect DNA from cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6) and John Cunningham virus (JCV) via real-time polymerase chain reaction (PCR).ResultsCryptococcal meningitis accounted for 63.0% (34 of 54) of all causes of meningitis, 13.0% (7/54) for TB, 9.3% (5/54) for Toxoplasma gondii. Of 54 samples sent for viral PCR, 31.5% (17/54) were positive, 12 (22.2%) for CMV, 2 (3.7%) for VZV, 1 (1.9%) for EBV, 1 (1.9%) for HHV-6 and 1 (1.9%) for JCV. No patient was positive for HSV. Pathogen-based treatment and high GCS score tended to have a lower mortality rate, whereas patients with multiple pathogens infection, seizures or intracranial hypertension showed higher odds of death.ConclusionCNS OIs are frequent and multiple pathogens often coexist in CSF. Cryptococcal meningitis is the most prevalent CNS disorders among AIDS. The utility of molecular diagnostics for pathogen identification combined with the knowledge provided by the investigation may improve the diagnosis of AIDS related OIs in resource-limited developing countries, but the cost-efficacy remains to be further evaluated.

A Rapid and Sensitive Method for Simultaneous Screening of Nine Foodborne Pathogens Using High-Performance Liquid Chromatography Assay

We developed a multiplex PCR-based procedure followed by high-performance liquid chromatography (mPCR-HPLC) assay for high-throughput screening foodborne pathogens, including Salmonella spp., Listeria monocytogenes, Enterobacter sakazakii, Staphylococcus aureus, Shigella spp., Escherichia coli O157:H7, Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus. Vibrio species-specific primers were designed targeting dnaJ gene, and pathogen-specific primers for L. monocytogenes, Salmonella spp., E. sakazakii, E. coli O157:H7, Shigella spp., and S. aureus were designed targeting iap, fimY, 16S ribosomal RNA (rRNA), rfbE, ipaH, and 442 genes, respectively. PCR products were analyzed using WAVE-4500 DNA system equipped with PS-DVB-C18 particles DNASep column and each specific amplicon generated a characteristic chromatographic profile. Detection limit of mPCR-HPLC assay was ca. 101 CFU/mL in pure cultures and less than 102 CFU/g in contaminated matrixes. A total of 395 bacterial strains were used for specificity testing of the mPCR-HPLC assay, and specific HPLC profiles were only produced in strains belonging to the target, showing a high specificity. Applying the assay to 2677 samples collected from clinical, food, and environmental sources revealed that 917 samples were positive, in accordance with bacterial isolation. The high sensitivity and specificity of mPCR-HPLC assay indicate its great potential to be a powerful tool for high-throughput screening foodborne pathogens.

Molecular detection of prokaryotes in apricot showing decline and leaf scorch symptoms in Iran

During field surveys in 2015 and early spring 2016 in orchards of Mahvelat and Khaf regions of Razavi-Khorassan province in Iran a severe apricot decline was observed in the 22 to 27% of the cultivars Shahroodi and Lasjerdi. Diseased trees exhibit generalized yellows, upward curling and scorch of the leaves, premature and small leaf, and often fruit dropping, together with growth reduction and premature leaf discoloration. Molecular detection carried out on extracted nucleic acid with PCR and pathogen specific primers allow to verify the presence of phytoplasmas belonging to ribosomal groups 16SrXII and 16SrI in mixed infection, and in some samples of bands referable to the presence of Xylella fastidiosa. The first detection of these pathogens in the same species represents a big threat not only for the Iranian apricot cultivations, but also for agriculture in general, considering that all the detected prokaryotes are infecting a high number of plant species.

Simultaneous detection of fungal, bacterial, and viral pathogens in insects by multiplex PCR and capillary electrophoresis

Beetles Protaetia brevitarsis seulensis Kolbe (Coleoptera: Cetoniidae) and Allomyrina dichotoma Linn. (Coleoptera: Scarabaeidae) are widely used in traditional medicine, and the number of insect-rearing farms is increasing in South Korea. The purpose of this study was to establish a multiplex PCR-based assay for rapid simultaneous detection of multiple pathogens causing insect diseases. Six insect parasites such as fungi Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) and Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae), bacteria Bacillus thuringiensis Berliner (Bacillales: Bacillaceae), Pseudomonas aeruginosa Migula (Pseudomonadales: Pseudomonadaceae), and Serratia marcescens Bizio (Enterobacteriales: Enterobacteriaceae), and Oryctes rhinoceros nudivirus were chosen based on the severity and incidence rate of insect diseases in South Korea. Pathogen-specific primers were designed and successfully applied for simultaneous detection of multiple infectious agents in farm-bred insects P. b. seulensis and A. dichotoma using multiplex PCR and high resolution capillary electrophoresis. Our results indicate that multiplex PCR is an effective and time-saving method for simultaneous detection of multiple infections in insects, and the QIAxcel capillary electrophoresis system is useful for quantitative evaluation of the individual impact of each infectious agent on the severity of insect disease. The approach designed in this study can be utilized for rapid and accurate diagnostics of infection in insect farms.

Guidelines for Selection of Tissues for Electron Microscopy Confirmation of Candidatus Liberibacter spp. in Huanglongbing-affected Citrus

Polymerase chain reaction (PCR) with the pathogen-specific primers and electron microscopy are the two techniques of choice that have been used for detection and identification of Candidatus Liberibacter spp. in the Huanglongbing (HLB)-affected citrus. Due to the low population and uneven distribution of Liberibacter in the diseased citrus trees finding the bacteria with transmission electron microscopy has been a challenge. Work with samples from HLB-affected citrus during the past 5 years has resulted in certain guidelines for the selection of plant tissues that have led to success in visualization of the bacteria. With PCR-positive field citrus trees, the best source is phloem tissue from seed coats of developing seeds in young fruit supported by leaves with blotchy mottle symptoms. These half mature seeds have been shown to contain large numbers of intact bacteria. However, when working with young potted trees there are usually no fruit to sample. In these trees we had some success in finding bacteria in petioles or midveins of PCR-positive, young developing leaves (2/3 to fully expanded, but not hardened) sampled above leaves showing blotchy mottle symptoms. However, we have found larger numbers of bacteria in roots of the potted diseased trees. We have experienced the most success in sampling root tissue in areas where the phloem has just completed differentiation, in PCR-positive small pioneer and fibrous roots showing primary and secondary growth.

A bead-based multiplex assay for the detection of DNA viruses infecting laboratory rodents.

The Federation of European Laboratory Animal Science Association (FELASA) recommends screening of laboratory rodents and biological materials for a broad variety of bacterial agents, viruses, and parasites. Methods commonly used to date for pathogen detection are neither cost-effective nor time- and animal-efficient or uniform. However, an infection even if silent alters experimental results through changing the animals’ physiology and increases inter-individual variability. As a consequence higher numbers of animals and experiments are needed for valid and significant results. We developed a novel high-throughput multiplex assay, called rodent DNA virus finder (rDVF) for the simultaneous identification of 24 DNA viruses infecting mice and rats. We detected all 24 DNA viruses with high specificity and reproducibility. Detection limits for the different DNA viruses varied between 10 and 1000 copies per PCR. The validation of rDVF was done with DNA isolated from homogenised organs amplified by pathogen specific primers in one multiplex PCR. The biotinylated amplicons were detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent Luminex beads. In conclusion, rDVF may have the potential to replace conventional testing and may simplify and improve routine detection of DNA viruses infecting rodents.