Conventional Polymerase Chain Reaction Tests Research Articles

Highly Sensitive Sars-Cov-2 Detection Using Au-Functionalized Laser-Induced Graphene Sensors

The 2019 coronavirus (COVID-19) pandemic has led to a drastic loss of human life and presented unprecedented challenges to global health, society, and the economy. Developing highly sensitive diagnostics for early detection of the infection is crucial in controlling the disease’s spread. Conventional polymerase chain reaction tests, while accurate, are relatively slow, expensive, and require trained personnel, causing strain on healthcare systems as they struggled to meet testing demands1. Electrochemical sensors offer advantages over traditional diagnostic methods including lower production cost, ease of use, real time analysis, and high sensitivity. Recently, laser-induced graphene (LIG) has gained significant attention in electrochemical sensing for its high surface area and excellent electron transfer properties. Various graphene modification methods have been used in developing electrochemical sensors, including Au-NP functionalization, choice of cross-linkers, and incubation parameters such as humidity, temperature, and duration; however, a systematic, concurrent comparison of these functionalization parameters is missing.In this work, through a comprehensive investigation of various functionalization steps, we developed electrochemical sensors based on Au-functionalized laser induced graphene (LIG) for detection of SARS-CoV-2 with an outstanding limit of detection (LOD) of 1.2 ag/mL. To optimize the sensors, we explored the functionalization of LIG with Au-NPs via direct laser processing. The porous structure of LIG increases the surface area available for electrochemical reactions and protein immobilization, enhancing its effectiveness for sensing applications2. We showed that functionalization of the working electrode LIG with Au-NPs improves the electrochemically active surface area by ~4.5-fold compared to the geometric area. We also investigated the impact of different crosslinkers and showed that PBA/Sulfo-NHS/EDC cross-linker improves the sensitivity and LOD significantly compared to PBASE. Additionally, we studied the effect of antigen mixing, showing that sensitivity and LOD improve by more than 1.6-fold and 5.5-fold using pipette mixing compared to static mixing. The incubation condition is also optimized. Moreover, the sensors exhibit excellent selectivity against other viruses, including HCoV-229E, HCoV-OC43, HCoV-NL63, and influenza H5N1. To show the diagnostic ability for analysis of real samples, the sensor performance in spiked saliva is also demonstrated.The insights gained from this study on graphene modification for SARS-CoV-2 detection may help optimize the fabrication of other graphene-based electrochemical sensors for detecting a variety of bioanalytes based on immobilization of biorecognition molecules. References Teymouri, “Recent advances and challenges of RT-PCR tests for the diagnosis of COVID-19,” Pathology - Research and Practice, vol. 221, p. 153443, 2021J. Lin et al., “Laser-induced porous graphene films from commercial polymers,” Nature Communications, vol. 5, no. 1, p. 5714,2014

RELATIONSHIP BETWEEN THE SPECTROMETRIC VALUES OF DNA, RNA, AND THE PCR PRESENCE OF A PATHOGEN IN SINGLE TICK SAMPLES

Aim. Ticks are the vectors of many pathogens, which cause diseases with fatal consequences. Polymerase chain reaction (PCR) was used to detect the presence of these different pathogens in ticks, but there is a need of isolated nucleic acid to conduct the molecular assays. In our previous research, we found that some ticks give huge yield of isolated nucleic acid during spectrometric measurements, therefore aim of this study is to find whether there is any relation between spectrometric values of DNA, RNA and presence of Borrelia burgdorferi as example pathogen in single tick samples. Methods. DNA and RNA were isolated with mini column method from single tick samples. They were run in real time as well as conventional PCR tests for the presence of Borrelia burgdorferi. The nucleic acid yields of isolated nucleic acid samples were measured with a spectrophotometer. Results. It was found that there were 47 ticks positive for Borrelia burgdorferi and 40 were negative. Average isolated DNA and RNA quantity was higher in pathogen positive ticks than those of negative ticks. There was no correlation between the yield of nucleic acid and presence of pathogen in a single tick, but there was tendency that pathogen positive tick gave higher yield of DNA and RNA during the isolation. Conclusions. This study shows some of Borrelia burgdorferi positive ticks give very high yield of DNA and RNA during the isolation. There is no correlation between presence of pathogen and nucleic acid in a single tick, but there is tendency that pathogen positive tick may have higher nucleic acid yield. Therefore, our recommendation is that laboratory should always measure the nucleic acid yield along with conducting the PCR tests.

Monkeypoxvirus (MPXV) in a Baby Monkey - Molecular Investigation

Background: Monkeypox is a zoonotic virus that transmits from animal to human and human to human. Despite the fact that the Monkeypox virus (MPXV) was initially isolated in 1958 and the first human case was reported in 1970, thousands of cases have been documented in European countries, the United States, and Turkey in recent years. The goal of this work was to present the results of the first molecular diagnostic analysis of MPXV-induced skin lesions in a baby monkey in Turkey. Case: In 2019, a baby monkey in Antalya Zoo developed skin lesions after being brought from Africa by a female monkey. The baby monkey died within a few weeks due to postnatal respiratory problems. Skin lesions were taken from this case and analyzed molecularly and virologically at the Department of Pathology, Burdur Mehmet Akif Ersoy University Faculty of Veterinary Medicine, and the Department of Virology. To perform molecular diagnosis, skin and pock lesions developed after inoculation on the chorioallantoic membrane (CAM) of the embryonated chicken eggs (ECE) were extracted, and 25 parapoxvirus (orf virus) type-specific primers and 2 MPXV type-specific primers were examined. The inoculum was prepared from the skin lesions for virus isolation, which was carried out in Vero cell culture. Then, the virus titer was determined using the microtitration method. The polymerase chain reaction (PCR) test with 25 parapoxvirus (orf virus) type-specific primers on the extracted skin lesion samples did not detect the presence of viral genome. The presence of viral genome was detected in 1 of the 2 MPXV type-specific primers acidophilic-type inclusion body (ATI gene) in the skin lesion extracts. However, the presence of viral genome could not be determined by the Gabon (1/2) primers. Cytopathological effects (CPE) were observed 72 h. after inoculating the skin lesion inoculum in Vero cells. The virus titer was determined to be 1022 TCID50 /mL. During immunohistochemical examination with orf virus antibodies, positive reaction was observed in the epidermal cells. Discussion: The researchers have been investigated the reservoir or natural hosts of MPXV. The virus has been found in squirrels, rodents, monkeys and chimpanzees. Multiple examinations have revealed that a number of animal species, primarily rodents and nonhuman primates, are susceptible to the virus. The presence of MPXV was detected in the skin lesion of a baby monkey in the study. Capripoxvirus, Cervidpoxvirus, Avipoxvirus, Molluscipoxvirus, Orthopoxvirus, Leporipoxvirus, Suipoxvirus, Yatapoxvirus, and Parapoxvirus are all members of the Chordopoxvirinae subgroup. However, no relationship between MPXV and parapoxvirus (25 type-specific primers) was found in the extracted skin lesion samples. Virus isolation, electron microscopy, (PCR), IgM and IgG ELISA, immunofluorescence assays, and histopathological examination are all laboratory diagnostic procedures that can be used to diagnose monkeypox infections. To detect the MPXV agent and/or specific viral DNA sequences, real-time or traditional PCR techniques should be utilized. Hemagglutinin, ATI gene, and the crmB gene are all MPXV genes which are commonly used for conventional PCR testing. A MPXV type-specific primer (ATI gene) observed the presence of viral genome in the skin lesion extract. After inoculating the skin lesion inoculum in Vero cells, (CPE) were observed. The virus titer was found to be extremely high. Positive reactions were seen in epidermal cells during immunohistochemical testing with orf virus antibodies. Keywords: Monkeypox virus, molecular diagnosis, virological diagnosis, immunohistochemistry, Parapoxvirus.

First case of mpox diagnosed in Queensland, Australia: clinical and molecular aspects.

First case of mpox diagnosed in Queensland, Australia: clinical and molecular aspects.

Evaluation of dry stored disposable sensor strip on rapid SARS-CoV-2 detection platform

This study investigated a SARS-CoV-2 virus detection mechanism using dry-stored disposable strips. The accuracy of this sensing platform is as good as polymerase chain reaction (PCR) with a detection time of fewer than 30 s. SARS-CoV-2 antibodies were biofunctionalized on disposable strips similar to glucose detection strips to detect the presence and concentrations of SARS-CoV-2 in saliva samples. Eight 1 ms electric pulses were sent through the sensor strip with a saliva sample in its microfluidic chamber. A circuit board embedded with MOSFET was also employed to amplify the detected signals and convert the signal to digital readings displayed on an LCD screen. The COV-antibody functionalized disposable strips were stored in a dry condition for at least one day before analyzing clinical human saliva samples with known cycling threshold (Ct) values confirmed with conventional PCR tests. Results demonstrate our system is capable of showing qualitative positive or negative results within 30 s and providing quantitative SARS-CoV-2 concentrations in terms of Ct values in 5 min.

Experimental In vitro Study to Assess the Antibacte-rial Activity of Thymus vulgaris Oil on Streptococ-cus Sanguinis

Background: The Streptococcus genus are the predominant bacteria in the mouth and the Streptococcus sanguinis is one of them which performing a primary function for expansion of dental biofilm. Gingival and periodontal disorders are caused by dental biofilm, today, there is a necessity to discover naturally presenting antibacterial compounds from herbs with less side effects as a substitutive to the commonly handled chlorohexidine. Thus, the purpose of this study was to assess the antibacterial activity of thymus vulgaris oil on Streptococcus sanguinis bacteria In vitro. Materials and Methods: Human supragingival plaque samples were taken from 10 subjects, then morphological and microscopical examination, biochemical tests, Optochin test, hemolytic ability test and conventional polymerase chain reaction test was applied to assure coincidence of Streptococcus sanguinis. The sensitivity of bacteria to Thymus vulgaris oil, the minimum concentration that inhibit the growth and killed the test bacteria were identified a partly as comparable to 0.2% chlorhexidine as a positive control and10 % dimethyl sulphoxide as a negative control. Results: Thymus vulgaris oil presented a significant antibacterial property on Streptococcus sanguinis with several grades of inhibition zones. When compared to Chlorohexidine, Thymus vulgaris oil had stronger antibacterial properties. The minimum concentration that inhibited the growth and killed Streptococcus sanguinis was (0.09%). Conclusion: Thymus vulgaris oil displayed higher antibacterial action with each concentration on Streptococcus sanguinis as compared to Chlorohexidine, therefore; it can be used as a natural substitutive oral health care product to Chlorohexidine.

Efecto del desbridamiento periodontal más azitromicina sistémica en sujetos con periodontitis estadio III: Un ensayo clínico controlado aleatorio

Aim: To evaluate the effect of the systemic administration of azi-thromycin (AZM) as an adjunct to non-surgical periodontal therapy (NSPT) on the clinical and microbiological variables of patients with periodontitis Material and Methods: Eighteen volunteers received NSPT combined with placebo or AZM (500 mg/day) for 3 days (n=9/group). They were monitored clinically for probing pocket depth (PPD), clinical attachment level (CAL), O'Leary index (OI), bleeding on probing (BoP) at baseline and during the first, third and sixth month and microbiologically, at baseline and at 3 and 6 months after therapy, by conventional polymerase chain reaction tests. Results: Fourteen patients completed the study (n=7/group). Differences statistically significant were observed among both groups. The experimental group presented: A PPD mean (p=0.04) significantly lower and PPD reduction (p=0.02), at 6-months post NSPT. Regarding changes (∆), at the third month post NSPT, there was a significant increase in the number of shallow sites (p<0.001) and a decrease in the intermediate sites (p<0.001). In addition, a significant decrease in the mean number of deep sites (p=0.04) was detected at 6 months post treatment. There was also a significant decrease in periodontal index BoP at 1 (p=0.01), 3 (p<0.001) and 6 (p=0.01) months and OI at 3- and 6-months (p<0.001), post treatment. Regarding the presence of periodontal pathogens, no significant differences were observed, intra and inter groups. Conclusion: AZM as an adjuvant to NSPT provides additional beneficial effects for PPD and BoP compared to NSPT alone.

The Impact of Rapid PCR Testing for Viral Respiratory Infections on Acute Admissions From the Emergency Department and Inpatient Length of Stay.

Diagnosing influenza and respiratory syncytial virus (RSV) with conventional multiplex respiratory viral polymerase chain reaction (PCR) takes 24-72 hours compared with under two hours for recently available rapid PCR tests. We investigated the impact of rapid diagnosis of acute respiratory viral infection on admission odds from the emergency department (ED) and acute inpatient length of stay (LOS), as well as concordance between the rapid and conventional PCR tests used at our institution. Single-center retrospective cohort study of patients presenting to the ED with influenza-like illness. We compared the odds of admission and acute LOS in patients investigated with rapid PCR, those investigated with conventional PCR, and those investigated with both tests. Multivariable logistic regression was used to assess the odds of admission, while linear regression was used to assess LOS. There was no significant change in the odds of admission among patients who received the rapid PCR compared to conventional PCR (odds ratio: 1.01, 95% confidence interval [CI]: 0.50-2.02; p = 0.96). There was also no significant difference in LOS of admitted patients who received rapid PCR testing (regression coefficient: -0.32, 95% CI: -1.75 to 1.12; p = 0.66). The rapid PCR test used at our institution yielded fully concordant results with conventional PCR testing. Rapid PCR testing is as sensitive as conventional PCR testing for the diagnosis of influenza and RSV but is neither associated with a significant impact on admission nor inpatient LOS. Further research is needed to assess the impact of rapid testing on isolation room use.

Molecular evidence of Leishmania spp. in spider monkeys (Ateles geoffroyi) from The Tuxtlas Biosphere Reserve, Veracruz, Mexico.

The black-handed spider monkey (Ateles geoffroyi) is a platyrrhine primate distributed in southern Mexico, Central America, and part of South America. Two subspecies inhabit Mexico: Ateles geoffroyi vellerosus and Ateles geoffroyi yucatanensis, both threatened with extinction. Serological evidence of exposure of spider monkeys to various groups of parasites such as Trypanosoma cruzi in México and Leishmania spp. in Brazil has been reported. The genus Leishmania encompasses about 23 species of flagellate protozoa that are transmitted by the bite of females of Phlebotominae sand flies. These parasites cause a zoonotic disease called leishmaniasis, which generates skin, mucocutaneous and/or visceral manifestations. The aim of the present study was to demonstrate the presence of Leishmania sp. in spider monkeys from the Tuxtlas Biosphere Reserve, Veracruz, Mexico. Blood samples from 10 free- ranging specimens of A. geoffroyi yucatanensis and 11 specimens in captivity of A. geoffroyi vellerosus were collected and used. The samples were subjected to a conventional Polymerase Chain Reaction test for the identification of a 116bp fragment of a region from the kinetoplast minicircle of the parasite. Our analyzes showed that 71.4% of the sampled animals had fragment sizes compatible with Leishmania spp. The implications involve the survival of the specimens and the possibility that these primates act as sentinels of the disease. Furthermore, it is the first report suggesting the presence of Leishmania spp. in A. geoffroyi vellerosus and A. geoffroyi yucatanensis in Veracruz, Mexico.

Virologic detection and molecular characterization of canine parvovirus-2 in dogs

This study aimed to search the extent of spread of various canine parvovirus variants in Egypt from year 2019 to year 2020 and the ability of the present used vaccines in the protection .In this study, twenty fecal samples were collected from diarrheic puppies. These samples were tested by rapid CPV Ag test kits for the detection of CPV-2 Ag. Ten samples were found positive for CPV2 by rapid Ag test kit (VDRG CPV Ag rapid kit).Positive samples were propagated on Vero cell culture for 8 passages, CPE appears in the form of cell rounding and detachment within 5 days. A conventional polymerase chain reaction test was performed on tissue culture isolates. Partial sequencing of five randomly selected positive samples revealed the presence of 2 CPV2C and 3 CPV2B.CPV2B represents the highest level of the variant serotype of CPV circulating in Egypt through records of egyptian isolated cases at the gene bank . One of the isolate has 100% identity with a CPV2B of cat origin. Our isolates CPVS1 and CPVS4 have 100% nucleotide identity with each other and with CPV cairo1-19 and CPV cairo3-19 belonging to CPV2C , this variant is also widespread all over the country. Continuous accurate molecular and epidemiological studies are needed to follow-up the new mutation of the virus genome which may result in vaccination failure(The majority of the used vaccines in Egypt are of CPV2 origin not of variant origin). The use of CPV2b based vaccine gave better protection than cpv2 based vaccine(Wide range protection against CPV2 variants). The obvious spread and emergence of newly introduced CPV variants at different Egyptian governorates as cited at previous study necessitates updated molecular based epidemiological studies of field isolates continuously to follow up the efficacy of the concurrent used vaccines.

ART Initiation for Infants Diagnosed With HIV Through Point of Care and Conventional Polymerase Chain Reaction Testing in Kenya: A Case Series.

We sought to understand the sequence of testing and treatment among nine infants offered both conventional and point-of-care testing and diagnosed as HIV-positive by 6 months of age in Kenya. One infant received per protocol testing and treatment. Patient-level (late presentation and disengagement), provider-level (reluctance and error/oversight) and system-level (stock outs, errors) challenges delayed diagnosis and treatment. Early point-of-care testing can streamline testing; however, challenges mitigate benefits.

Use of Point-of-Care Nucleic Acid Tests Beyond Early Infant Diagnosis of HIV: A Retrospective Case Review in Lesotho.

Rapid diagnostic tests (RDTs) for HIV antibodies remain the primary method of diagnosis of HIV in individuals over age 18 months in Lesotho. Although antibody tests have high sensitivity and specificity, up to 2.3% of serial two-test algorithms can have discrepant results between RDTs. In the case of inconclusive RDT results, Lesotho guidelines at the time of this study recommended either repeat testing with the same RDT algorithm after 14 days or immediately collect a blood sample to be sent for laboratory-based polymerase chain reaction testing. Point-of-care qualitative nucleic acid tests (POC qual NAT) may have benefits in rapidly resolving these inconclusive results, particularly when compared with repeating RDTs later or conventional polymerase chain reaction testing at the National Reference Laboratory. Hospitals and clinics at 29 locations throughout Lesotho that had access to point-of-care nucleic acid testing. Retrospective case review. We identified 100 testing records where POC qual NAT was used to resolve inconclusive RDTs per Lesotho guidelines. Eighty-nine percent of patients received their results in a median of one day from their inconclusive RDT result (interquartile range 0-7 days). Sixty-eight patients (68%) were determined to be HIV positive based on POC nucleic acid tests (NATs), of which 54 (79%) were started on antiretroviral therapy (ART). Median time from inconclusive RDT result to initiation of ART therapy was 2 days (interquartile range 0-14 days). Three patients in this review were pregnant at the time of testing; one was HIV positive by POC qual NAT and was started on ART therapy the same day. As the availability of POC qual NAT platforms increases, they may serve as feasible options for rapid resolution of inconclusive results and initiation of ART, particularly in populations with high risk of imminent transmission.

Developing a Pre-Testing Diagnostic Tool for COVID-19 Using Big Data Predictive Analytics

Background: Standard Polymerase Chain reaction (PCR) tests for SARS-COV-2 are in short supply to meet demand in many countries presenting a need to improve testing efficiency. Pre-testing tools can be used to ensure continued public safety as systems move through the pandemic. In this study we set out to create an instrument based on big data predictive tools to assess pre-test probability for COVID-19. Methods: We analyzed data reported by the Israeli Ministry of Health (IMOH) for standard PCR tests done for SARS-COV-2 from March to April, 2020, overall 108,852 cases. Demographics and symptoms of the patients were collected at time of testing. Four supervised machine learning algorithms were used to analyze 20,537 test results of cases who presented with symptoms. Model results were used to develop efficient pre-test diagnostic tool. Findings: Of symptomatic patients tested, 6,427 (31.3%) tested positive for SARS-COV-2, and 14,110 (68.7%) tested negative. In all models used headache, shortness of breath, sore throat, fever, and having contact with an infected person came up as most predictive of a positive test. The area under the curve of the receiver operating characteristic curve for the test sample was found to be 0.88 and the misclassification rate was between 4.7% and 6.5% for all predictive models, demonstrating effective classification ability. Using our pre-test probability screening tool with conventional PCR testing can potentially increase efficiency by 141%. Interpretation: We suggest a simple diagnostic pre-test tool for assessing the probability of infection can increase efficiency of testing and effectiveness of public health COVID-19 programs. Funding: NoneDeclaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The authors noted that the analysis presented was approved by the IMOH Data Sharing Institutional Review Board.

A Resposta das Tecnologias de Saúde à Pandemia de COVID-19: Uma Luz ao Fundo do Túnel?

The responses to the threats of the COVID-19 pandemic have been based on public health measures that prevent the spread of infection. After months of intense international research, the first vaccines against COVID-19 have already begun to be approved and administered. The first vaccine against COVID-19 was administered in Portugal, on December 27th, 2020, as it happened in all European counterparts. In addition to vaccines, the efficacy and safety of several medicines in the treatment of COVID-19 are still being studied, some of them already approved with other therapeutic indications. The need to generate information that guarantees the safety of the medicines under study, underlines the fundamental role of the pharmacovigilance system in every process. Finally, over time, both the use of tests and the use of medical devices associated with COVID-19 have also evolved very quickly. In testing, in addition to conventional PCR (polymerase chain reaction), rapid antibody and antigen tests have emerged. On the other hand, medical devices and personal protective equipment whose availability, in the first phase, was a serious problem, are now widely available, with emphasis on the greater national offer and technical improvements.

Accuracy of polimerase chain reaction for the diagnosis of pleural tuberculosis

Polymerase chain reaction (PCR)-based techniques to detect Mycobacterium tuberculosis DNA in respiratory specimens have been increasingly used to diagnose pulmonary tuberculosis. Their use in non-respiratory specimens to diagnose extrapulmonary tuberculosis is, however, controversial. In this study, we estimated the accuracy of three in-country commercialized PCR-based diagnostic techniques in pleural fluid samples for the diagnosis of pleural tuberculosis. Patients underwent thoracenthesis for diagnosis purposes; pleural fluid aliquots were frozen and subsequently submitted to two real time PCR tests (COBAS(®)TAQMAN(®)MTB and Xpert(®)MTB/Rif) and one conventional PCR test (Detect-TB(®)). Two different reference standards were considered: probable tuberculosis (based on clinical grounds) and confirmed tuberculosis (bacteriologically or histologically). Ninety-three patients were included, of whom 65 with pleural tuberculosis, 35 of them confirmed. Sensitivities were 29% for COBAS(®)TAQMAN(®)MTB, 3% for Xpert(®)MTB/Rif and 3% for Detect-TB(®); specificities were 86%, 100% and 97% respectively, considering confirmed tuberculosis. Considering all cases, sensitivities were 16%, 3% and 2%, and specificities, 86%, 100%, and 97%. Compared to the 95% sensitivity of adenosine deaminase, the most sensitive test for pleural tuberculosis, the sensitivities of the three PCR-based tests were very low. We conclude that at present, there is no major place for such tests in routine clinical use.

Molecular study on selected vector-borne infections in urban stray colony cats in northern Italy.

Feline vector-borne diseases can be caused by a range of pathogens transmitted by arthropods. Many of these infections have zoonotic implications, and stray cats are potential sentinels for human and pet health. This study investigated the prevalence of selected vector-borne infections in stray colony cats in Milan. Blood samples from 260 stray cats were evaluated, using conventional polymerase chain reaction tests (cPCRs), for the presence of DNA associated with Rickettsia species, Anaplasma phagocytophilum and Ehrlichia species. Positive cPCR results occurred in 127/260 subjects (48.9%; 95% confidence interval [CI] = 40.7-58.1), with a prevalence of 31.9% (83/260, 95% CI = 25.4-39.6) for Rickettsia species, 17.7% (46/260, 95% CI= 13.0-23.6) for A phagocytophilum, and 5.4% (14/260, 95% CI = 2.9-9.0) for Ehrlichia species. There was no statistical association between a positive PCR test for vector-borne infections surveyed and colony location, age, gender, body condition score or complete blood count abnormalities, nor feline immunodeficiency virus, feline leukaemia virus or Toxoplasma gondii status. The only variable linked to positive PCR results was detection of signs of ocular infection and PCR positivity for Rickettsia species (P = 0.04, odds ratio [OR] = 2.2, 95% CI = 1.1-4.4, P = 0.02). There is a significant prevalence of vector-borne infections with zoonotic potential in urban stray cats in Milan. Thus, dogs and pet cats with outdoor access should be monitored and treated for ectoparasites on a regular basis to minimise risks of disease and the potential transmission of zoonotic agents to people.

Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae

Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.

The Enhanced Pneumococcal LAMP Assay: A Clinical Tool for the Diagnosis of Meningitis Due to Streptococcus pneumoniae

Background Streptococcus pneumoniae is a leading cause of invasive bacterial disease in developed and developing countries. We studied the loop-mediated isothermal amplification (LAMP) technique to assess its suitability for detecting S. pneumoniae nucleic acid in cerebrospinal fluid (CSF).Methodology/Principal FindingsWe established an improved LAMP assay targeting the lytA gene (Streptococcus pneumoniae [Sp] LAMP). The analytical specificity of the primers was validated by using 32 reference strains (10 Streptococcus and seven non-Streptococcus species) plus 25 clinical alpha-hemolytic streptococcal strains, including four S. pneumoniae strains and 21 other strains (3 S. oralis, 17 S. mitis, and one Streptococcus species) harboring virulence factor-encoding genes (lytA or ply). Within 30 minutes, the assay could detect as few as 10 copies of both purified DNA and spiked CSF specimens with greater sensitivity than conventional polymerase chain reaction (PCR). The linear determination range for this assay is 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the clinical sensitivity and specificity of the Sp LAMP assay using 106 randomly selected CSF specimens from children with suspected meningitis in Korea, China and Vietnam. For comparison, CSF specimens were also tested against conventional PCR and culture tests. The detection rate of the LAMP method was substantially higher than the rates of PCR and culture tests. In this small sample, relative to the LAMP assay, the clinical sensitivity of PCR and culture tests was 54.5% and 33.3%, respectively, while clinical specificity of the two tests was 100%.Conclusions/SignificanceCompared to PCR, Sp LAMP detected S. pneumoniae with higher analytical and clinical sensitivity. This specific and sensitive LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.

Direct simultaneous detection of 6 sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis

The availability of a reliable and user-friendly method to identify pathogens causing sexually transmitted diseases (STDs) is essential to reduce the complications and spread of infection. In this study, genital/urinary specimens from 113 patients with STDs were simultaneously tested for 6 pathogens using the automated Seeplex® (Seegene, Seoul, Korea) multiplex polymerase chain reaction (PCR)-based STD6B auto-capillary electrophoresis (ACE) system. The results were compared with conventional reference methods, including culture and PCR tests. The sensitivity of STD6B ACE was found to be 100% for Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Neisseria gonorrhoeae, and Trichomonas vaginalis, and 98% for genital Ureoplasma (U. urealyticum and U. parvum). Specificity ranged from 97% to 100%. One pathogen was detected in 51 specimens, and 2 or more pathogens were detected in 24. In conclusion, the multiplex PCR and ACE system is highly sensitive and specific for the rapid, simultaneous detection of STD pathogens directly from a single specimen.

Sensitive Detection of Fusarium circinatum in Pine Seed by Combining an Enrichment Procedure with a Real-Time Polymerase Chain Reaction Using Dual-Labeled Probe Chemistry

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer-probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.