Duplex PCR Research Articles

PM 7/121 (2) ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’

Specific scopeThis Standard describes a diagnostic protocol for ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’.1This Standard should be used in conjunction with PM 7/76 (5) Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2014–09. Revised in 2021–04.

تعیین جنسیت گوزن قرمز (زیرگونه مرال) با استفاده از تکنیک Duplex PCR به عنوان، روشی مناسب برای تخمین اندازه مؤثر جمعیت، جهت طراحی برنامه های حفاظت ژنتیکی آن در زیستگاه های کشور

Unfortunately, due to the small population size and isolation of red deer (Cervus elaphus) in protected area-Arasbaran-Aynalu, there are inbreeding and the highly rate of bottleneck. Sex controlling for sex maintain effective population size as appropriate, may reduce inbreeding. For this purpose the objective of this study was access and optimization techniques for Sex determination in Red deer(Ceruvus elaphus); suitable method for estimating the effective population size for genetic conservation programs in Arasbaran and the country habitats. For this purpose, bleeding in deer, taken from jugular vein and tail vein after anesthesia, blood samples in tubes containing EDTA vaccum with ice -40c to the gen bank of Department of East Azerbaijan was transferred. Duplex PCR using primers based on the chromosome (Y) in accordance with the sex-determining gene DEAD-box Y-linked protein (DBY) simultaneously used with BMC1009 locus were amplified as an internal control per each reaction. The results are reliable, there are two bands 280-310 (polymorphic band) and 180 base pairs (bp) in male deer and 280-310 (polymorphic band) base pairs (bp) in female deer showed. To confirm this, residual PCR products was applied for sequencing. Genotyping results showed that 8 females and 15 males were identified as indicators of effective population size 4, respectively.

Detection of cytomegalovirus in plasma of patient’s HIV by duplex real time PCR

Cytomegalovirus (CMV) is able to cause serious and even deadly diseases in immunocompromised patients. It is important to have a sensitive, specific and molecular viral tests for its detection, using as targets, key genes for viral replication. The following genes have been used in the molecular detection of CMV: UL122 (replication) and UL83 (most abundant protein of the tegument). Detect and quantify CMV, by real-time duplex PCR, from a minimum amount of plasma. The UL122 and UL83 genes were amplified with different fluorophores, by real-time duplex PCR. To quantify CMV, curves were generated, starting with DNA-CMV (1.0-0.0000001 ng). The dynamic range of “master” duplex straight had a pendent (m) −3.0, the amplification efficiency was 115.44% plasmas from patients with HIV viral load ≥ 100,000 copies/mL, 11.36% were true positive for CMV and 88.64% had no amplifications or they were outside of the linear range of molecular detection. This test identified two important CMV genes (UL122 and UL83) in a single reaction (FAM:VIC), viral detection was confirmed from a minimum amount of plasma. This mean a smaller amount of biological sample required and would add a tool to the clinical area, as well as a lower consumption of reagents and materials.

Rapid detection of phenotypes Bombay sedel and nonsecretor rs200157007 SNP (302C\u2009>\u2009T) by real-time PCR-based methods

The sedel allele is one of the nonsecretor alleles (se) of FUT2 generated by an Alu-mediated recombination event and was first found in Indian Bombay phenotype individuals who have anti-H, anti-A, and anti-B antibodies in their serum. As well as anti-A, and anti-B antibodies, anti-H is clinically significant because it causes sever hemolytic transfusion reactions. Like sedel, se302 having a missense single nucleotide polymorphism (SNP), 302C > T, is characteristic of South Asians with a frequency of 10–30%. We developed a real-time PCR melting curve analysis for detection of sedel using a 127-bp amplicon encompassing the breakpoint junction. In addition, by performing duplex PCR by amplifying a 65-bp amplicon of the FUT2 coding region at the same time, we could determine the zygosity of sedel in a single tube. We also developed an Eprobe-mediated PCR assay (Eprobe-PCR) for detection of 302C > T of FUT2. These methods were validated by analyzing 58 Tamils and 54 Sinhalese in Sri Lanka. Both the duplex PCR melting curve analysis for determination of sedel zygosity and the Eprobe-PCR assay for detection of 302C > T exactly determined three genotypes. In addition, the results of the present methods were in complete agreement with those obtained by previously established methods. The two present methods were reliable and seem to be advantageous for large-scale association studies of FUT2 polymorphisms in South Asian populations.

Parasitological and molecular search for Leishmania natural infection in phlebotomine sand flies in peri-urban and rural sites of an Argentinean area endemic for tegumentary leishmaniasis

Leishmaniases are neglected tropical diseases caused by Leishmania spp. parasites transmitted by the bite of phlebotomine sand flies. In Argentina, the most endemic area of American tegumentary leishmaniasis (ATL) has been Orán department, Province of Salta, where Leishmania (Viannia) braziliensis prevails and Nyssomyia neivai is considered as its vector, although there is no accurate and sufficient information in this regard. The aim of this work was to search for natural infection by Leishmania spp. in sand flies from peri-urban and rural sites with ATL background in Orán department. For this, sand flies were caught at five sites; female sand flies captured with Shannon trap were dissected to microscopically examine their gut contents, while females captured with CDC traps were molecularly analyzed by duplex PCR with two primer pairs to simultaneously amplify kinetoplast DNA (kDNA) and mammalian actin. A total of 1921 females were captured, with Ny. neivai being the most abundant species (89%), followed by Migonemyia migonei (6%) and cortelezzii complex (3%). No natural infection was found in any of them neither by dissection nor by PCR, although the detection limit of kDNA PCR was up to 25 promastigotes. The absence of infected females in peri-urban sites suggest that the transmission did not take place in those environments during the study period. Future searches for natural infection should focus on rural settings to deepen knowledge and elucidate the role of the circulating sand fly species as all have been linked to ATL transmission at other sites.

Detecção de β-lactamase, blaZ e mecA em amostras de Staphylococcus aureus resistentes à penicilina isoladas de mastite bovina em Garanhuns, Brasil

There are few reports in the literature about genetic determinants of resistance to β-lactams in Staphylococcus aureusisolated from dairy cattle located in the municipality of Garanhuns, state of Pernambuco, Brazil. Thus, this study aimed to investigate the production of β-lactamase and the presence of the blaZ and mecA genes in penicillin-resistant S. aureus isolated from cases of subclinical bovine mastitis in the city of Garanhuns. Forty-six strains of penicillin-resistant S. aureus were evalu-ated using the nitrocefin disc test and duplex PCR. The results revealed that 45 strains (97.8%) were positive for β-lactamase production and 44 (95.7%) carried the blaZ gene. Among the latter, 43 (97.7%) were β-lactamase producers and only one (2.3%) was not. The mecA gene was not detected in any of the isolates investigated. The results suggest that enzymatic inacti-vation is the main β-lactam resistance mechanism expressed by S. aureus in the herds analyzed.

Identification of the respiratory tract infection due to methicillin-resistant Staphylococcus aureus by TaqMan real-time PCR

Abstract The methicillin-resistant Staphylococcus aureus (MRSA) is a significant human pathogenic bacterium that is endemic within hospitals around the world. The identification and inspection of MRSA in clinical samples is quite helpful both in advising individual patients about the required care and in tracking these species. The goal of this study was to present a modern, faster, and more accurate diagnostic technique to operate on the real-time duplex PCR applicable to S. aureus/MRSA monitoring in Iraqi patients. For this reason, the S. aureus-specific nuc gene sequence and the mecA gene sequence were checked simultaneously. To estimate the assay efficiency, a set of six target strains, 34 non-target strains, and 296 clinical specimens were used. The findings obtained from the diagnosis of a total of 296 isolates based on phenotypic characteristics and biochemical tests showed that 146 (49.32%) were classified as individuals with respiratory tract infections of S. aureus with a total male to female ratio of 1.47, and 142 isolates demonstrated methicillin resistance. 142 MRSA isolates were investigated in the molecular analysis, all MRSA isolates had positive results for the nuc gene and 138 isolates were positive for the mecA gene. The current real-time PCR assay has 97% sensitivity, 100% specificity, and 98% accuracy. Running title: Identification of the MRSA by real time PCR

A new duplex PCR assay for the rapid screening of mating-type idiomorphs of pathogenic Sporothrix species

Sporothrix schenckii and allied species are thermodimorphic fungi widely distributed in nature which causes human and animal sporotrichosis, the most common subcutaneous mycosis globally. Sporotrichosis is acquired after a traumatic inoculation of soil or plant material contaminated with Sporothrix propagules or through bites and scratches from diseased cats. In Ascomycota, the master regulators of sex are MAT genes that lie in a single mating-type locus, in Sporothrix these are determined by two nonhomologous alleles, MAT1-1 and MAT1-2. We assessed the whole-genome sequences of medically relevant Sporothrix to develop a single-tube duplex PCR assay to screen S. brasiliensis, S. schenckii, S. globosa, and S. luriei idiomorphs (MAT1-1 or MAT1-2) and understand the distribution and incidence of mating-type strains from natural populations. Using our duplex PCR assay, a 673 bp amplicon (α-box protein) was consistently amplified from all MAT1-1 isolates, while a 291 bp fragment was only amplified from the isolates harboring MAT1-2 (HMG box). Molecular evidence suggests heterothallism (self-sterility) as the unique mating strategy among the species evaluated. The mating-type identity of 93 isolates revealed a nearly equal distribution (1:1 ratio) of mating type alleles within species but deviating between different outbreak areas. Remarkably, for S. brasiliensis in Rio de Janeiro, we report an overwhelming occurrence of MAT1-2 (1:13 ratio; χ2 = 10.286, P = 0.0013) opposing the high prevalence MAT1-1 in the Rio Grande do Sul (10:1 ratio; χ2 = 7.364, P = 0.0067). Therefore, the population structure of Sporothrix species refers from paucity to regular cycles of sexual recombination in most of the studied regions. Our PCR-based mating-type diagnostic assay is proposed here as an important marker to track the geographical expansion during the long-lasting outbreak of cat-transmitted sporotrichosis driven by S. brasiliensis.

Prevalence, virulence factor and antimicrobial resistance analysis of Salmonella Enteritidis from poultry and egg samples in Iran

BackgroundSalmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most common serovars, associated with human salmonellosis. The food-borne outbreak of this bacterium is mainly related to the consumption of contaminated poultry meat and poultry products, including eggs. Therefore, rapid and accurate detection, besides investigation of virulence characteristics and antimicrobial resistance profiles of S. Enteritidis in poultry and poultry egg samples is essential. A total of 3125 samples (2250 poultry and 875 poultry egg samples), sent to the administrative centers of veterinary microbiology laboratories in six provinces of Iran, were examined for Salmonella contamination, according to the ISO 6579 guideline. Next, duplex PCR was conducted on 250 presumptive Salmonella isolates to detect invA gene for identification of the genus Salmonella and sdf gene for identification of S. Enteritidis. Subsequently, the S. Enteritidis isolates were examined for detection of important virulence genes (pagC, cdtB, msgA, spaN, tolC, lpfC, and spvC) and determination of antibiotic resistance patterns against nalidixic acid, trimethoprim-sulfamethoxazole, cephalothin, ceftazidime, colistin sulfate, and kanamycin by the disk diffusion method.ResultsOverall, 8.7 and 2.3% of poultry samples and 6.3 and 1.3% of eggs were contaminated with Salmonella species and S. Enteritidis, respectively. The invA and msgA genes (100%) and cdtB gene (6.3%) had the highest and the lowest prevalence rates in S. Enteritidis isolates. The spvC gene, which is mainly located on the Salmonella virulence plasmid, was detected in 50.8% of S. Enteritidis isolates. The S. Enteritidis isolates showed the highest and the lowest resistance to nalidixic acid (87.3%) and ceftazidime (11.1%), respectively. Unfortunately, 27.0% of S. Enteritidis isolates were multidrug-resistant (MDR).ConclusionThe rate of contamination with Salmonella in the poultry and egg samples, besides the presence of antimicrobial resistant and MDR Salmonella isolates harboring the virulence genes in these samples, could significantly affect food safety and subsequently, human health. Therefore, continuous monitoring of animal-source foods, enhancement of poultry farm control measures, and limiting the use of antibiotics for prophylactic purposes in food producing animals, are essential for reducing the zoonotic risk of this foodborne pathogen for consumers and also choosing effective antibiotics for the treatment of salmonellosis.

Development and assessment of a duplex droplet digital PCR method for quantification of GM rice Kemingdao.

The implementation of genetically modified organism (GMO) labeling policies requires accurate quantitative methods to measure the GMO content in test samples. A Kemingdao/phospholipase D (KMD/PLD) duplex ddPCR method was established with rice genomic DNA (gDNA) of homozygous KMD as template by optimizing the annealing temperature and cycle number. Duplex ddPCR showed a linear response over the dynamic range from 68 to 175,000 copies, covering four orders of magnitude. The limit of detection (LOD) and limit of quantification (LOQ) for duplex ddPCR were determined to be 9 copies and 34 copies of the rice haploid genome, respectively. A very high dilution factor would result in unacceptable bias and coefficients of variation for determining copy number of the gDNA solution, and more than 1000 copies of the DNA template in one reaction is preferred to obtain accurate quantitative results by duplex PCR. Five blinded DNA samples with copy number ratio of 10%, 5%, 1%, 0.1%, and 0.05%, and three blinded real-life matrix samples with mass fraction of 5%, 1%, and 0.5% were quantified by duplex ddPCR, simplex ddPCR, and qPCR. These three methods all gave comparable GMO content and copy numbers within the required precision, but the duplex ddPCR showed the narrowest uncertainty interval and provided the highest precision in comparison to simplex ddPCR and qPCR. The ddPCR is a more appealing and reliable technology for the accurate quantification of GMO content than simplex ddPCR and qPCR considering the uncertainty and precision of quantitative results, the time consumption of generating droplets, and the cost of ddPCR reagents.

Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates

BackgroundCampylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates.MethodsA total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping.ResultsOut of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates.ConclusionSignificant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

Two new rapid PCR-based methods for identification of Acinetobacter baumannii isolated from clinical samples

The accurate identification of Acinetobacter spp. is challenging due to their high phenotypic and biochemical similarities. Because clinical relevance and antibiotic susceptibility are significantly different among different genomic species of Acinetobacter, the exact identification of A. baumannii is necessary and it can help us prevent inappropriate antibiotic use and inferior clinical care. This project employed a sequence-specific PCR assay for the rpoB region in A. baumannii to distinguish it from non-Acinetobacter baumannii Acinetobacter species. Moreover, a duplex PCR assay was used to detect blaOXA-51-like and gluconolactonase genes as a second identification method. In this study, 210 isolates of Acinetobacter spp. were considered and identified by PCR-sequencing of rpoB gene as a reference test. PCR-sequencing of rpoB revealed that 179 isolates were A. baumannii and 31 were non- A. baumannii Acinetobacter strains. PCR amplification targeting the rpoB gene as the first method, detected 182 isolates of A. baumannii, while duplex PCR assay confirmed 163 isolates as A. baumannii. Data analysis indicated that the sensitivities of sequence-specific PCR of the rpoB gene and duplex PCR assay were 100% and 91.06%, respectively, while specificities were 91.18% and 100%, respectively. Given the data, it was revealed that these two methods showed a reasonable potential for the accurate identification of A. baumannnii from non- A. baumannii species. Sequence-specific PCR assay for the rpoB gene and duplex PCR assay for blaOXA-51-like and gluconolactonase genes are rapid, reliable and cost-effective methods which can be used in clinical laboratories for the accurate identification of A. baumannii.

Deteksi Salmonella spp. pada Telur Ayam Konsumsi dari Peternakan Ayam Ras dan Pasar Tradisional di Bali

This study aims to early detect Salmonella spp. and identify serotypes in commercial chicken eggs from layer chicken farms and traditional markets in Bali. Early detection study of Salmonella spp. was carried out by conventional bacteriological methods, while serotype identification by duplex Polymerase Chain Reaction (d-PCR) test against the invA gene from Salmonella spp. and the sefA gene from Salmonella enteritidis. Egg samples in this study were taken from 10 layer chicken farms in Bali which included districts of Bangli, Gianyar, Tabanan and Karangasem. Egg samples from traditional markets were taken from 18 traditional markets from the districts of Bangli, Gianyar, Tabanan, Karangasem, Badung, and Denpasar City. Samples were eggshells and egg whites. Analysis of positive results from Salmonella spp. described descriptively. The results showed that eggshells and white eggs from all of the layer chicken farms are negative contaminated with Salmonella spp. (0%). In eggshell samples taken from the traditional markets of Taman Bali and Tulikup from the districts of Bangli and Gianyar, positive with Salmonella spp. (11,1%) by conventional bacteriological tests. In the duplex Polymerase Chain Reaction test, S. enteritidis serotypes were identified. The finding contamination of Salmonella enteritidis in commercial chicken eggs from traditional markets require periodically detection to prevent the occurrence of salmonellosis due to consumption of contaminated chicken eggs in traditional markets in Bali.

Detection of species adulteration in meat products and Mozzarella-type cheeses using duplex PCR of mitochondrial cyt b gene: A food safety concern in Bangladesh

Food origin authentication is imperative for consumer protection. This study explored meat and milk species identification as a pioneering country-specific report on mislabeling prevalence among processed meat and milk products in Bangladesh. Meat products (64; sausages, burger patty, meatball, kabab) labeled as chicken or beef and Mozzarella-type cheeses (25), made in Bangladesh and of overseas origins, were analyzed for species detection. Two duplex PCR (cattle-buffalo and chicken-pig) were applied with species-specific mitochondrial Cyt b gene primers and determine to be accurate for species identification in meat and milk. Bangladeshi origin beef-labeled products were found to be mixed with buffalo (n = 2) and chicken (n = 5) suggesting up to one third of products might be mislabeled. Such mislabeling would allow these ‘beef’ products to charge a higher price compared with chicken products that command a lower price. Imported meat products were also found mislabeled with buffalo and chicken. Cheese samples, declared as bovine, were found to contain buffalo DNA, and no cattle or buffalo DNA was found in six imported cheese samples. All the meat and cheese products were Halal, as none contained pig DNA. This pilot study shows the majority of products were labelled correctly, but large proportions were not and strict monitoring is recommended to ensure food safety and address consumer preferences, especially religious concerns.

Identification and Authentication of Commercial Mi-iuy Croaker (Miichthys miiuy) Products by Two PCR-Based Methods

Mi-iuy croaker (Miichthys miiuy) is one of the most important ingredients of Korean cuisine and, thus, has a high economic value. However, the similar morphological traits among croaker fish belonging to family Sciaenidae are often exploited for seafood fraud. In this study, an M. miiuy-specific primer set was designed and further improved by the development of a rapid and cost-effective duplex PCR method. The specificity of M. miiuy-specific duplex PCR was tested using 22 seafood species, and no cross-reactivity was observed. The sensitivity of the PCR assay was found to be 0.1 ng/μL. For the first time, labeling compliance of 43 commercial mi-iuy croaker products was verified using both full DNA barcoding and M. miiuy-specific duplex PCR methods. For species identification, BOLDSYSTEMS and GenBank database were screened with the consensus sequences of each PCR product as a query. This identification result was further confirmed using the M. miiuy-specific duplex PCR method. The findings of this study revealed that principal species substituted were law croaker (Pseudotolithus senegallus, n = 4), bigeye croaker (Micropogonias megalops, n = 3), whitemouth croaker (Micropogonias furnieri, n = 1), and tigertoothed croaker (Otolithes ruber, n = 1). A significant percentage (21%) of mislabeling was present in commercial mi-iuy products sold on the South Korean market.

A New Duplex PCR-Assay for the Detection and Identification of Paracoccidioides Species.

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the P. brasiliensis complex and P. lutzii in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for Paracoccidioides species. Primers PbraCx-F and PbraCx-R targeting P. brasiliensis DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting P. lutzii DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 Paracoccidioides revealed 100% specificity (AUC = 1.000, 95%CI 0.972–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the P. brasiliensis complex (n = 7) or P. lutzii (n = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient’s organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to P. brasiliensis (S1) and P. lutzii in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the P. brasiliensis complex and P. lutzii, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas.

Development of a simple and cost-effective gel-based duplex PCR method to identify both encapsulated and unencapsulated Neisseria meningitidis applicable under resource-limited conditions

Tens of thousands of cases of invasive meningococcal diseases (IMD) with thousands of deaths are reported annually worldwide; however, only approximately 40 cases occur each year in Japan. Therefore, the majority of medical technologists in Japan have never performed or prepared for analyses of the causative agent, Neisseria meningitidis. Since IMD outbreaks have been reported at mass gathering events, the risk of IMD will increase in Japan in 2021 because of the Olympics. In the present study, we developed a new simple gel-based duplex PCR method that may be employed by the majority Japanese clinical laboratories. It is simple to perform and time- and cost-effectively identifies encapsulated and unencapsulated N. meningitidis by detecting the encapsulated N. meningitidis-specific ctrB and N. meningitidis-specific ggt genes. We consider this simple and cost-effective identification method to compensate for the lack of experience and resource-poor conditions in most Japanese laboratories in which N. meningitidis has rarely been examined.

Detection and Frequency of Enterotoxin (cpa,cpe) Genes of Clostridium perfringens Isolated from Dehydrated Vegetables by PCR

Background and Objective: Clostridium perfringens is an anaerobic bacterium, commonly present in retail foods. Its enterotoxin-producing ability, short generation time, ability to grow at elevated temperatures, and spore-forming ability, allows it to survive in food-processing temperatures, and cause foodborne illness. The aim of study was to screen dehydrated vegetables contaminated with cpe and cpa carrying C.perfringens. Materials and Methods: This is descriptive-analytical study, was carried out on 140 samples (70 unpacked and 70 packed) dehydrated vegetables collected from different areas of Tehran. Samples were inoculated on peptone and sulfite polymyxin sulfadiazine (SPS) agar for enrichment. The enrichment culture was then incubated on anaerobic condition for 48 hours. The black colonies were selected for identification test and PCR. The bacterial colonies were identified by biochemical tests, and duplex PCR was performed for α-toxin (cpa) and enterotoxin (cpe) genes. Results: In general 13 samples (9.3) were identified as C. perfringens using phenotypic methods, all of the isolates were also positive for cpa but negative for cpe gene. The contamination rate for packed vegetables was 12.8 and for unpacked was 5.7. Conclusion: Our finding showed that contamination of packed dehydrated vegetables was higher than unpacked; this might be due to drying as well as packaging process. We found that these isolates were negative for enterotoxin.

Validación de una técnica de PCR dúplex usando el gen E y RNasa P para el diagnóstico de SARS-CoV-2

IntroductionReverse transcriptase - polymerase chain reaction (RT-PCR) is the standard technique for SARS-CoV-2 diagnosis. The World Health Organization recommends the Charité-Berlin protocol for COVID-19 diagnosis, which requires triple PCR, limiting the process capability of laboratories and delaying the results. In order to reduce these limitations, a duplex PCR is validated for the detection of the E and RNase P genes. MethodsWe compared the limit of detection, sensitivity and specificity of the duplex PCR technique (E gene and RNase P) against the monoplex standard (E gene) in RNA samples from a SARS-CoV-2 isolate and 88 clinical specimens with previously known results. The repeatability and reproducibility of the threshold cycle values (Ct) were determined in two independent laboratories of the Faculty of Medicine of the Universidad de Antioquia, using different reagents and real time instruments. ResultsThere were no significant differences in the Ct results between both techniques (p = 0.84). Using the monoplex PCR of E gene as a reference, the interrater reliability analysis showed similarity between the two techniques, with a kappa coefficient of 0.89, the sensitivity and the specificity of duplex PCR were 90% and 87%, respectively. ConclusionsDuplex PCR does not affect the sensitivity and specificity reported by the Charité, Berlin protocol, being a useful tool for SARS-CoV-2 screening in clinical samples.

First report of pathogenic Leptospira spp. in Tadarida brasiliensis bats (family Molossidae) and Eptesicus furinalis (family Vespertilionidae) of Argentina. New host species in this country?

Leptospirosis is an endemic disease caused by Leptospira spp., a bacterium that affects animals and humans. In recent years, the number of reports of leptospirosis in wild animals has increased, which highlights the need to study the infectious agents in these animals. In this study, a duplex PCR for the detection of leptospiral DNA was performed on 50 kidney samples from bats, and a MAT (Microscopic Agglutination Test) for serological detection of anti-leptospiral antibodies was applied to 47 serum samples from bats from different regions of Buenos Aires Province, Argentina.DNA was extracted using Chelex-100 and duplex PCR was performed by targeting the detection of genes secY and flaB, of pathogenic Leptospira spp. Of the 50 kidney samples, 3 were positive for Eumops sp. and Tadarida brasiliensis by duplex PCR. Of the 47 serum samples, 12 were positive for different serovars: Leptospira interrogans serovars Icterohaemorrhagiae, Cynopteri and Bataviae, and Leptospira borgpetersenii serovar Ballum. This is the first report of the detection of pathogenic leptospires by serology in bats belonging to the T. brasiliensis and Eptesicus furinalis species in Argentina. In addition, this is the first report of the detection of pathogenic leptospiral DNA by PCR in T. brasiliensis species. The detection of Leptospira spp. in these wild animals shows that they may play an important role as wildlife reservoirs of leptospires.