PCR-based Molecular Methods Research Articles

Evaluation of a rapid Loop Mediated Isothermal Amplification (LAMP) test for the laboratory diagnosis of sexually transmitted infections.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

Development of a specific and sensitive diagnostic PCR for rapid molecular authentication of the medicinal plant Portulaca oleracea

Adulteration by Bacopa monnieri (BM) in Portulaca oleracea (PO) plants frequently occurs; it decreases the efficacy of traditional Chinese medicine (TCM) and leads to fraud in the herbal marketplace. In this study, a diagnostic PCR assay was established for the rapid authentication of PO and BM in the herbal market. The sequence divergences in internal transcribed spacer 2 (ITS2) between PO and its adulterant species were used to design diagnostic PCR primers. The specific designed primer sets were evaluated and show that the diagnostic PCR assay can be used to verify the authenticity of PO and BM. The detection limits of the primer set for PO and BM identification were 10 pg and 1 pg, respectively. The reactivity of diagnostic PCR was 0.1% PO genomic DNA and 0.01% BM genomic DNA in the test sample during DNA amplification. In addition, multiplex PCR (mPCR) for PO and BM identification was also established. The samples were more susceptible to the effect of steaming in authentication by singleplex PCR and mPCR than boiling and drying treatment. Furthermore, commercial samples from the market were used to demonstrate the applicability of the developed diagnostic PCR for PO authentication and diagnose BM adulteration, and the investigation found that approximately 72.2% (13/18) of PO plants in the herbal market were adulterated. In conclusion, the diagnostic PCR assay was successfully developed and its specificity, sensitivity and reactivity for PO and BM authentication were proven. These developed PCR-based molecular methods can be applied as an identification tool for PO authenticity and can be practically applied for inspection of BM adulteration in the herbal market in the future.

Development of species-specific PCR detection for three Mycogone species causing wet bubble disease in white button mushroom

Agaricus bisporus, commonly known as white button mushroom, is a major agricultural crop worldwide. However, wet bubble disease in Agaricus bisporus, caused by three Mycogone species, namely Mycogone perniciosa, M. rosea and M. xinjiangensis, leads to tremendous economic losses in China. The three species are difficult to distinguish based on morphology and pathogenicity. An early, fast and accurate method to detect and differentiate among these species is essential for controlling the disease. Based on differences in Tef-1α gene sequences, therefore, three sets of species-specific primers, T-6-3/AT-4, T-2/AT-2 and T-7-1/AT-3, were successfully designed for the detection and identification of M. perniciosa, M. rosea and M. xinjiangensis, respectively, using an endpoint PCR assay. Subsequently, the specificities of the three primers were demonstrated using 43 isolates, including Mycogone spp., A. bisporus, Trichoderma harzianum, Trichothecium roseum, Cladobotryum dendroides, Lecanicillium fungicola, Fusarium solani and other soil-borne pathogens. The detection limits for M. perniciosa, M. rosea and M. xinjiangensis were 0.5 pg/μL, 0.2 ng/μL and 20 pg/μL of DNA template, respectively, suggesting strong amplification with the three primer pairs. Moreover, the species-specific primers efficiently detecteerd the target species in artificially inoculated mushrooms and soils. This is the first report to identify and distinguish each of the three Mycogone species. Besides, this PCR-based molecular method could provide a sensitive, quick and highly specific tool to discriminate among the three Mycogone species. Collectively, the assay developed in this study could have great application value for early detection of Mycogone species in soil and effective control of wet bubble disease.

Probiotic Characterization of Arsenic-resistant Lactic Acid Bacteria for Possible Application as Arsenic Bioremediation Tool in Fish for Safe Fish Food Production.

Arsenic (As) contamination of water and food is a global problem posing a severe threat to environmental and human health; therefore, fish as an aquatic animal is immensely affected by the hazardous impacts of As. The present study aimed to explore the As-resistant probiotic bacteria and characterize their potential for applying as an As bioremediation tool in fish. As-resistant lactic acid bacteria (LAB) were isolated from sludge samples of an old stabilization pond/lagoon of wastewater treatment plant using spared plate techniques. The potential probiotic was selected by assessing the sequential probiotic characterization, As resistance and removal properties. The selected probiotic was identified by PCR-based molecular method using 16S rDNA. A total of 51 As-resistant LAB were isolated from sludge samples. Potential six As-resistant LAB strains (As4, 11, 20, 21, 41 and 48) were selected from 51 isolates through sequential probiotic characterizations using mimic fish gastrointestinal conditions. The selected probiotics displayed relatively elevated As (> 1000mg L-1), cadmium (20-100mg L-1) and lead (> 2000mg L-1)-resistant patterns and excellent As-removal efficiencies (0.0012-0.0044mg As mg cell-1h-1) from water along with favourable various associative probiotic properties. The 16S rDNA sequence-based molecular identification and phylogenetic analysis revealed that the strains As4, 11, 20, 21, 41 and 48 belong to Limosilactobacillus fermentum (Lactobacillus fermentum according to old taxonomy). The As removal and survival in mimic gastrointestinal conditions of fish indicated that new Limosilactobacillus fermentum strains could be employed as the novel and potential probiotic tools for possible bioremediation of As and other pollutants in the fish to prevent the bioaccumulation and toxicity impacts of As in fish for cleaner and safe fish food production.

Potential Application of PCR Based Molecular Methods in Fish Pathogen Identification: A Review

Molecular biology developments have led to fast growth in new methods for fish disease diagnosis. Molecular diagnostic methods are rapid and more specific, more sensitive than the culture of pathogens, serology, histology, and biochemical methods which are traditionally utilized to identify causative agent fish disease. Molecular diagnostic methods are valuable for detecting specific pathogens that are difficult to culture in vitro or require a long cultivation period and it significantly more rapid in providing results compared to culture. It enables earlier informed decision-making and rapid diagnosis of bacteremia, particularly for low levels of bacteria in specimens. Molecular techniques which have the major significance are mainly PCR-based molecular diagnostic methods including Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Multiplex Polymerase Chain Reaction (multiplexPCR), and Random Amplified Polymorphic DNA (RAPD). These have been increasingly utilized to diagnose fish disease for the last recent years. Molecular diagnostic methods can detect pathogens from asymptomatic fish, so disease outbreaks could be prevented. As a consequence, antibiotic treatment can be reduced and the development of antibiotic-resistant bacteria can be eliminated. In this review paper, we attempt to summarize the potentiality of PCR-based molecular diagnostic methods and their application in fish pathogen identification.

Association between SP-A rs1965708 gene polymorphism and allergic rhinitis risk in Chinese population.

BackgroundPulmonary surfactant protein A (SP‐A) in the respiratory tract plays an important role in host. In the present, we assessed the association between SP‐A gene polymorphism and allergic rhinitis.MethodsUsing a case–control design, we compared the genotype frequencies of SP‐A rs1965708 between allergic rhinitis patients and healthy control group. Genotyping was performed using real‐time quantitative PCR‐based molecular identification methods. Univariate and multivariate logistic regression were performed to quantitatively assess the association between rs1965708 polymorphism and allergic rhinitis, and the odds ratio (OR) and 95% confidence interval (CI) were also calculated.Results500 patients with allergic rhinitis and 500 healthy controls were included in the study. Compared with the CC genotype, we found that AA genotype of rs1965708 could increase the allergic rhinitis risk in the univariate analysis (OR = 2.63, 95% CI: 1.56–4.54, p = 0.000). For dominant model, we found no significant difference in the dominant model (OR = 1.14, 95% CI: 0.86–1.52, p = 0.367). In the recessive model, the CC genotype could elevate the risk of allergic rhinitis compared with CC + AA genotype (OR = 2.70, 95% CI: 1.61–4.54, p = 0.000). Similar results were also found in the allele model (OR = 1.28, 95% CI: 1.07–1.54, p = 0.008). Interactions between rs1965708 AA or AC and smoking increased the allergic rhinitis risk.ConclusionsThe rs1965708 variants of SP‐A gene polymorphism are associated with allergic rhinitis, and the A allele could increase the allergic rhinitis risk. The AA SNP variants that interact with smoking may alter the susceptibility to allergic rhinitis.

The temporal evaluation of RAS and BRAF mutation by liquid biopsy at progression after bevacizumab combinations in patients with metastatic colorectal cancer (mCRC).

e15587 Background: Expanded RAS analysis is essential for the selection of biologic agents in mCRC. RAS mutations indicates anti-EGFR unresponsiveness. In this study, we aimed to investigate RAS and BRAF mutations by liquid biopsy at progression in patients with RAS mutant mCRC. Methods: Sixty patients with mCRC who harbored tissue RAS mutations were prospectively analyzed between July 2019 and April 2020. All the patients treated with chemotherapy plus bevacizumab combinations . The plasma samples of the patients were analyzed after progression of bevacizumab combinations. RAS mutation profile was evaluated in plasma using Idylla PCR-based molecular diagnostics method, which enables rapid detection of common mutations in RAS and BRAF genes in circulating tumor DNA (ctDNA). Kaplan-Meier method was used for survival analysis and log-rank test was performed for comparison of groups. Results: The median age of the patients was 60 years (IQR:35-83 years) and female was (n=23, 38.3%). Primary tumor was located in the left colon in 81.7% of all patients. There were 95.0% KRAS and 5% NRAS mutations in baseline tissue biopsy. As a result of liquid biopsy after progression, 55.0% of the patients had KRAS, 3.3% NRAS and 3.3% had BRAF mutations. The RAS mutation detected in 58.3% of the patients. While there was no significant difference in terms of clinicopathological features between wild type (RAS/BRAF) and mutant type (RAS/BRAF) determined by liquid biopsy, the overall survival (OS) of the wild type group was significantly longer than mutant group (43.8 vs. 20.4 months, p= 0.002). Conclusions: This study demonstrated that there may be changes in RAS/BRAF mutation from plasma analysis after progression in patients with mCRC. Since better survival in the patient group with wild type was detected compared to the RAS concordance group, the evaluation of RAS mutation status at the time of progression may be important in terms of disease prognosis and treatment options.

Effects of Long-Term Exposure to Increased Salinity on the Amphibian Skin Bacterium Erwinia toletana.

Amphibian's skin bacterial community may help them to cope with several types of environmental perturbations, including osmotic stress caused by increased salinity. This work assessed whether an amphibian skin bacterium could increase its tolerance to NaCl after a long-term exposure to this salt. A strain of Erwinia toletana, isolated from the skin of Pelophylax perezi, was exposed to two salinity scenarios (with 18g/L of NaCl): (1) long-term exposure (for 46days; Et-NaCl), and (2) long-term exposure followed by a recovery period (exposure for 30days to NaCl and then to LB medium for 16days; Et-R). After exposure, the sensitivity of E. toletana clonal populations to NaCl was assessed by exposing them to 6 NaCl concentrations (LB medium spiked with NaCl) plus a control (LB medium). Genotypic alterations were assessed by PCR-based molecular typing method (BOX-PCR). The results showed that tolerance of E. toletana to NaCl slightly increased after the long-term exposure, EC50 for growth were: 22.5g/L (8.64-36.4) for Et-LB; 30.3g/L (23.2-37.4) for Et-NaCl; and 26.1g/L (19.332.9) for Et-R. Differences in metabolic activity were observed between Et-LB and Et-R and between Et-NaCl and Et-R, suggesting the use of different substrates by this bacterium when exposed to salinized environments. NaCl-induced genotypic alterations were not detected. This work suggests that E. toletana exposed to low levels of salinity, activate different metabolic pathways to cope with osmotic stress. These findings may be further explored to be used in bioaugmentation procedures through the supplementation with this bacterium of the skin microbiome of natural populations of amphibians exposed to salinization.

A novel method for the detection and diagnosis of virus infections in honey bees

In terms of infectious diseases caused by a variety of microorganisms, the ability to promptly and accurately identify the causative agents is the first step on the path to all types of effective management of such infections. Among the various factors that are affecting global bee health, viruses have often been linked to honey bee colony losses and they pose a serious threat to the fraction of agriculture that depends on the service of pollinators. Over the past few decades, PCR-based molecular methods have provided powerful tools for rapid, specific, and sensitive detection and the quantification of difficult-to-grow pathogenic microorganisms such as viruses in honey bees. However, PCR-based methods require nucleic acid extraction and purification, which can be quite laborious and time-consuming and they involve the use of organic solvents and chaotropic agents like phenol and chloroform which are volatile and highly toxic.In response, we developed a novel and non-sacrificial method for detecting viral infections in honey bees. As little as 1 μl of hemolymph was collected from adult workers, larvae, and queens of bee colonies by puncturing the soft inter-tergal integument between the second and third dorsal tergum with a fine glass capillary. The hemolymph was then diluted and subjected to RT-PCR analysis directly. The puncture wound caused by the glass capillary was found to heal automatically and rapidly without any trouble and the lifespan of the experimental workers remained unaffected. Using this method, we detected multiple viruses including Deformed wing virus (DWV), Black queen cell virus (BQCV), and Sacbrood virus (SBV) in infected bees. Furthermore, expressed transcripts that indicate the induction of innate immune response to the virus infections were also detected in the hemolymph of infected bees. The simplicity and cost-effectiveness of this innovative approach will allow it to be a valuable, time-saving, safer, and more environmentally friendly contribution to bee disease management programs.

Loop-Mediated Isothermal Amplification in Schistosomiasis.

Human schistosomiasis is one of the most important parasitic diseases, causing around 250 million cases (mostly in Africa) and 280,000–500,000 deaths every year. Due to the limited resources and the far-removed nature of many endemic areas, the implementation of new, sensitive and specific diagnostic tools has had little success. This is particularly true for PCR-based molecular methods that require expensive equipment and trained personnel to be executed. Loop-mediated isothermal amplification (LAMP) along with other isothermal techniques appeared in the early 21st century as an alternative to those methods, overcoming some of the aforementioned limitations and achieving a more inexpensive diagnostic. However, to this date, neither LAMP nor any other isothermal technique have signified a meaningful change in the way schistosomiasis diagnosis is routinely performed. Here, we present the recent developments in LAMP-based schistosomiasis diagnosis. We expose the main advantages and disadvantages of LAMP technology over PCR and other classical diagnostic methods focusing in various research approaches on intermediate hosts, animal models and patients. We also examine its potential clinical application in post-therapy monitoring, as well as its usefulness as a point-of-care test.

Diagnostic performance between histopathological and molecular methods in the detection of Helicobacter pylori

ABSTRACT Introduction: Helicobacter pylori (H. pylori) is a Gram negative bacterium considered to be the etiologic agent of various gastric diseases. The prevalence of bacterial infection varies according to age, geographic location, ethnicity and socioeconomic status. The chronic infection caused by microorganism can favor the development of severe pathologies such as gastric adenocarcinoma. In this sense, early diagnosis is essential for a better prognosis and therapeutic success. Several diagnostic methods performed using invasive and non-invasive techniques, with different sensitivity and specificity, have been used in the detection of H. pylori. Objective: To compare the performance of the molecular and histopathological technique used in the diagnosis of H. pylori infection. Methods: 76 gastric tissue samples were collected from dyspeptic patients who underwent molecular and histopathological diagnosis. Molecular detection was performed using the ribosomal gene (16S rRNA) using the polymerase chain reaction (PCR) technique. Results: The PCR-based molecular diagnostic method detected the bacterium in 63.1% of the samples, while the histopathological test identified the microorganism in only 38.1% of gastric biopsies. The data demonstrated that the PCR technique was about 1.6 times more sensitive than the histopathological technique. Conclusion: The PCR technique was the most efficient diagnostic method for detecting H. pylori and can be implemented in the laboratory routine as a complementary test for the early detection of H. pylori.

Genome-wide assessment and development of molecular diagnostic methods for imidacloprid-resistance in the brown planthopper, Nilaparvata lugens (Hemiptera; Delphacidae).

The brown planthopper, Nilaparvata lugens (Stål), is one of the most notorious pests of rice throughout Asia. The brown planthopper has developed high resistance to imidacloprid, a member of neonicotinoid insecticides. Several genes and mutations conferring imidacloprid resistance in N. lugens, especially in eastern and southeastern Asia populations, have been reported. Thus, the key mechanisms of imidacloprid resistance need to be examined. RNA-seq analyses revealed that only one cytochrome P450 monooxygenase gene, CYP6ER1, was commonly upregulated in the five resistant strains tested. Sequences of CYP6ER1, which were highly expressed in the imidacloprid-resistant strains, contained a three-nucleotide deletion in the coding region, and amino acid substitutions and deletion, compared to that in an imidacloprid-susceptible strain. RNAi-mediated gene knockdown of CYP6ER1 increased imidacloprid susceptibility in a resistant strain. Further, we established two simple and convenient PCR-based molecular diagnostic methods to detect the CYP6ER1 locus with the three-nucleotide deletion. Using these methods, the resistance of F2 progenies derived from the crosses of F1 siblings from susceptible and resistant parents was analyzed, showing that the imidacloprid resistance had a relationship to the CYP6ER1 locus with the three-nucleotide deletion. The overexpression of a variant CYP6ER1 with amino acid substitutions and deletion was involved in imidacloprid resistance in N. lugens. Based on these findings, molecular diagnostic methods have been developed and are promising tools for monitoring imidacloprid resistance in paddy fields. © 2020 Society of Chemical Industry.

Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

Cyanoprokaryotes biodiversity in nine shallow Bulgarian wetlands

The results from the investigation of the phytoplankton in nine shallow wetlands, situated in Central and Eastern Bulgaria, conducted in the frame of recent projects related to identifying harmful algal blooms and algal toxins in the country are demonstrated. Therefore, the focus is on the cyanoprokaryotes and their toxigenic species in particular. The species were identified using conventional light microscopy, while for distinguishing the toxigenic species and strains PCR-based molecular methods were applied. Cyanoprokaryotes comprised about 30% of the total phytoplankton biodiversity of the studied wetlands and were unevenly spread among them. The presence and spread of toxigenic strains in the studied wetlands also did not show any strong geographical pattern. Taxonomic problems related with the evaluation of the real cyanoprokaryotic biodiversity and potential alien or invasive character of the morphospecies are discussed.

Molecular characterization of Blastocystis in cattle in Turkey.

Blastocystis genus exist in a wide variety of hosts, including humans, birds, insects, annelids, amphibians, fish, and mammals. PCR-based molecular diagnostic methods have been successfully used to detect Blastocystis spp. in feces, and small subunit ribosomal ribonucleic acid (SSU rRNA) gene-based subtyping is the preferred method for diagnosis. There has been discussion about the subtypes of Blastocystis spp. which has been detected so far. To date, 26 different subtypes have been reported. The aim of this study was to determine the existence and diversity of Blastocystis spp. in cattle. In our study, a total of 80 stool samples were collected from cows and calves at 13 different farms in Burdur and one farm in Aydın. Using molecular method, a total of 9 samples out of 80 samples were found to be positive (11.25%) for Blastocystis. As a result of sequence analysis of Blastocystis positive samples, the subtype 14 was detected on seven samples, while in the other two samples, Blastocystis subtype 10 was identified. The ST10 and ST14 subtypes are commonly reported in animals but not isolated from human. Our analyses showed genetic differences among Blastocystis subtypes. Our study is the first Blastocystis subtyping study from cattle in Turkey.

GENETIC SURVEY OF INVASIVE S. PNEUMONIAE SEROTYPES IN BULGARIA FOR A 5-YEAR PERIOD

Streptococcus pneumoniae colonises the mucosal lining of the upper respiratory tract and is an important cause of invasive infections affecting young children, adults over 65 years of age, the immunocompromised and individuals with chronic diseases. Recent studies have shown variations in virulence based on the high rate of pneumococcal recombination. PCR-based molecular methods are highly sensitive, specific and are becoming the preferred tool for quick and accurate diagnosis of bacterial meningitis which is required to be defined within 2-3 hours.
 During the 5-year survey period (2013-2017), 202 materials received as cerebrospinal fluid samples and pneumococcal strains isolated from patients diagnosed with meningitis, were examined by Real-time PCR in the reference laboratory at NCIPD. Serotyping of S. pneumoniae-positive materials was performed with conventional multiplex PCR and Real-time PCR with primers for 41serotypes/serogroups.
 There is a high incidence of S. pneumoniae serotypes not covered by the pneumococcal conjugate vaccine (PCV10) currently used in Bulgaria. It was found that all cases of meningitis caused by S. pneumoniae vaccine serotypes occurred in patients that were not vaccinated.

Rapid and visual detection of Group B streptococcus using recombinase polymerase amplification combined with lateral flow strips

Conventional culture method for detecting Group B streptococcus (GBS), a common pathogen of neonatal meningitis and sepsis, is time-consuming and unsensitive. Even though real-time fluorescence PCR-based molecular method is more accurate, it need special instrument and elaborate protocol. Here, we established a novel molecular method combining recombinase polymerase amplification with lateral flow strips for detecting GBS. The cAMP factor (cfb) gene is a highly specific and sensitive biomarker to identify GBS and is detectable by using 100 genomic copies as the amplification template. Clinical performance of this assay was evaluated by testing 130 samples, in comparison with culture method and real-time fluorescence PCR, and the results achieved 100% accuracy, which were the same with those of real-time fluorescence PCR, and were better than those of culture method with false-negative detection. This study provides a rapid and visual method, with clinical potential, for the detection of GBS infection of patients.

Detection of Salmonella enterica subsp. enterica Serovar Cubana from Naturally Contaminated Chick Feed

Because some significant outbreaks of human salmonellosis have been traced to contaminated animal feed, the rapid and efficient detection of Salmonella in feed is essential. However, the current U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) culture method that uses lactose broth as a preenrichment medium has not reliably supported the results of real-time PCR assays for certain foods. We evaluated the BAM culture method and a quantitative real-time PCR (qPCR) assay using two preenrichment media, modified buffered peptone water and lactose broth, to detect Salmonella enterica subsp. enterica serovar Cubana in naturally contaminated chick feed. After 24 h of incubation, the qPCR method was as sensitive as the culture method when modified buffered peptone water was used as the preenrichment medium but less sensitive than culture when lactose broth was used. After 48 h of incubation, detection of Salmonella Cubana by qPCR and by culture in either preenrichment medium was equivalent. We also compared the performance of the traditional serotyping method, which uses pure cultures of Salmonella grown on blood agar, to two molecular serotyping methods. The serotyping method based on whole genome sequencing also requires pure cultures, but the PCR-based molecular serotyping method can be done directly with the enriched culture medium. The PCR-based molecular serotyping method provided simple and rapid detection and identification of Salmonella Cubana. However, whole genome sequencing allows accurate identification of many Salmonella serotypes and highlights variations in the genomes, even in tight genomic clusters. We also compared the genome of the chick feed isolate with 58 Salmonella Cubana strains in GenBank and found that the chick feed isolate was very closely related to an isolate from a foodborne outbreak involving alfalfa sprouts.

Protist Communities in Moored Long-Term Sediment Traps (Fram Strait, Arctic)–Preservation with Mercury Chloride Allows for PCR-Based Molecular Genetic Analyses

Here we present a pilot study demonstrating, that preservation with mercury chloride allows the application of PCR-based molecular methods for the characterization of marine protist communities collected with moored long-term sediment traps. They can provide information on pelagic protist communities by collecting sinking plankton from the upper water column all year-round, even in remote polar oceans. Assessment of small protist species from the nano- and picoplankton fractions in sedimented material by microscopy is extremely challenging or almost impossible. Hence, comprehensive studies of variability in protist community composition in moored long-term sediment traps are scarce. Considering that marine nano- and picoeukaryotes are ecologically very important, new approaches are urgently needed to investigate protists in the smallest size-fractions of moored long-term sediment trap samples. We applied the quick and cost-effective Terminal Restriction Length Polymorphism (T-RFLP) on a set of selected samples that were collected between 2000 and 2010 in September at a depth of ~ 300 m in the area of the “LTER (Long-Term Ecological Research) site HAUSGARTEN“ in the eastern Fram Strait (Arctic). The results of these analyses suggest a change in the trapped protist community after 2002 in this area. A comparison of 18S sequences obtained via 454-pyrosequencing from samples collected in the water column and mercury chloride preserved sediment traps in 2009 and 2010 suggests, that sediment traps might reflect the pelagic eukaryotic microbial biodiversity qualitatively. Furthermore, we have indication that preservation with mercury chloride does not severely change the nucleotide composition of 18S rRNA genes in long-term sediment traps. Overall, we suggest that preservation with mercury chloride is a key to open the door for molecular genetic analyses of long-term sediment trap samples, and that PCR-based molecular methods have a strong potential to become an important tool for comprehensive taxonomic analyses of protist- and bacterial communities in moored long-term sediment traps.

ISSR-Derived Species-Specific SCAR Marker for Rapid and Accurate Authentication of Ocimum tenuiflorum L.

Ocimum tenuiflorum has been widely used in traditional medicine and has high medicinal value. High volume trade of this potential medicinal plant species led to unscrupulous adulteration of both crude drugs as well as formulations. Morphology-based authentication is difficult in cases of incomplete or damaged samples and in dried herbal materials. In such cases, PCR-based molecular methods may aid in accurate identification. The present study aimed at developing species-specific DNA marker(s) for the authentication of O. tenuiflorum. A species-specific amplicon (279 bp) generated through an inter-simple sequence repeat marker (UBC 835) in all individuals of O. tenuiflorum was cloned, sequenced, and a primer pair was developed (designated as CIM-OT-835F/CIM-OT-835R). The newly developed sequence characterized amplified region marker was validated through PCR amplification in all available seven species of Ocimum, and its specificity for O. tenuiflorum was confirmed with the consistent generation of an amplicon of 177 bp. The developed marker can be used for accurate and rapid identification of the species for certification purposes and will be useful in quality control of medicinal preparations containing this important medicinal species.