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Background: Leptospirosis is a potentially life-threatening disease caused by bacteria of the genus Leptospira. The accurate identification and characterization of Leptospira species are critical for disease surveillance, outbreak investigation, and treatment strategies. Molecular techniques, such as Polymerase Chain Reaction (PCR) and Deoxyribonucleic acid (DNA) sequencing, have revolutionized the field of microbiology, providing rapid and accurate identification of Leptospira strains. However, optimizing these molecular techniques for accurate examination of Leptospira species can be challenging due to the genetic diversity and complexity of these bacteria. Purpose: This research aims to identify the most suitable primers for the precise identification of pathogenic Leptospira strains. Method: This research used the PCR method, using LipL32, rrs2, seqY, LipL41, IcdA, and Adk primers. A total of 17 isolates of pathogenic Leptospira bacteria were cultured from Institute of Vector Control and Reservoir Disease (IVRCD) in Salatiga, Indonesia. Result: The results of the research showed that the LipL41 and IcdA primers were found to be effective in distinguishing pathogenic strains, while the seqY, LipL32, Adk, and rrs2 primers required further refinement. The suitable Melting Temperature (TM) or annealing temperature is 58°C with 35 cycles of amplification. DNA concentration and purity had an A260/A280 ratio ranging between 1.8 and 2.8. Conclusion: LipL41 (500 bp) and IcdA (700 bp) are suitable primers for identifying pathogenic Leptospira.

ABSTRACT Since the discovery of complete ammonia oxidizers (comammox) within the genus Nitrospira, their distribution and abundance across habitats have been intensively studied to better understand their ecological significance. Many primers targeting their ammonia monooxygenase subunit A gene (amoA) have been designed to detect and quantify comammox bacteria and to describe their community structure. We identified 38 published primers, but only few had high coverage and specificity for all known comammox Nitrospira or one of the two described subclades. For each target group, we comprehensively evaluated selected primer pairs using in silico analyses, endpoint PCRs, qPCRs, and amplicon sequencing on samples from various environments. Endpoint PCRs and qPCRs showed that the most commonly used primer pairs (comaA-244F/659R, comaB-244F/659R, and Ntsp-amoA162F/359R) produced several bands, which likely inflated quantifications via qPCR. In contrast, the recently published primer combinations CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R resulted mostly in a single band. Furthermore, amplicon sequencing demonstrated that these primer combinations also captured the highest richness of comammox Nitrospira. Taken together, our results indicate that few existing comammox amoA primer combinations have both high specificity and coverage and that the choice of these high-specificity and high-coverage primer pairs substantially impacts the accurate detection, quantification, and community description of comammox bacteria. We, therefore, recommend using the CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R primer pairs.IMPORTANCEBacteria that can fully convert ammonia via nitrite to nitrate, the complete ammonia oxidizers (comammox), were recently discovered and are found in many natural and engineered environments. PCR-based tools to study their abundance and diversity were rapidly developed, resulting in a plethora of primers available, many of which are widely used. The presence of comammox bacteria in an environment can, however, only be correctly determined if the used primers detect all members of this group while not detecting any other guilds. This study assesses the coverage and specificity of existing primers targeting comammox bacteria using both computational and standard molecular techniques, revealing large differences in their performance. The uniform usage of well-performing primers across studies could aid in generating comparable and generalizable data to better understand the importance of comammox bacteria in the environment.

Simple Summary: A lot of avian species are morphologically monomorphic, and thus, sex determination is difficult if it is based only on their external examination. Molecular sex identification has become a crucial tool in various fields of biology, including ornithology. This non-invasive method used to determine the genetic sex of birds of any age, in our case, Eudromia elegans (Paleognathae, Tinamiformes), can proceed with regard to the differences between avian male (ZZ) and female (ZW) chromosomes. According to our experience, the other molecular methods failed in the sex determination of this exact species. The new test was designed to be reliable, fast, and simple to determine the sex of the individuals of E. elegans. Molecular sexing has significant implications for understanding various aspects of its biology, including mating systems, population genetics, and conservation strategies. By accurately determining the sex of individuals within a population, researchers can investigate sex ratio patterns, reproductive success, and gene flow. Sex determination based just on morphological traits such as plumage dichromatism, sexual size dimorphism, behavior, or vocalizations is really challenging because of the sexual monomorphism present in more than half of avian species. Currently, a lot of them can be tested through DNA-based procedures, but they do not fit all the avian species, such as Eudromia elegans. The aim of this study was to design a new molecular method suitable for routine sex determination for that species that is fast, simple, and cost- and time- effective. DNA was isolated from dry blood stain and/or chest feather samples of E. elegans species. We used two sets of sex-specific primers (ZF/ZR and WF/WR) to amplify the expected fragments localized on the highly conserved CHD1 gene to distinguish between sexes due to the W-specific DNA sequence present only in females. We confirmed the accuracy and consistency of the PCR-based method based on length differences to distinguish between the sexes of E. elegans, which amplified two fragments in females and one fragment in males. [ABSTRACT FROM AUTHOR]

The major malaria mosquitoes, Anopheles gambiae s.l and Anopheles funestus, are some of the most studied organisms in medical research and also some of the most genetically diverse. When designing polymerase chain reaction (PCR) or hybridisation-based molecular assays, reliable primer and probe design is crucial. However, single nucleotide polymorphisms (SNPs) in primer binding sites can prevent primer binding, leading to null alleles, or bind suboptimally, leading to preferential amplification of specific alleles. Given the extreme genetic diversity of Anopheles mosquitoes, researchers need to consider this genetic variation when designing primers and probes to avoid amplification problems. In this note, we present a Python package, AnoPrimer, which exploits the Ag1000G and Af1000 datasets and allows users to rapidly design primers in An. gambiae or An. funestus, whilst summarising genetic variation in the primer binding sites and visualising the position of primer pairs. AnoPrimer allows the design of both genomic DNA and cDNA primers and hybridisation probes. By coupling this Python package with Google Colaboratory, AnoPrimer is an open and accessible platform for primer and probe design, hosted in the cloud for free. AnoPrimer is available here https://github.com/sanjaynagi/AnoPrimer and we hope it will be a useful resource for the community to design probe and primer sets that can be reliably deployed across the An. gambiae and funestus species ranges.

Microbes produce various types of volatile organic compounds (VOCs) through metabolism, which can be used for diagnostic purposes. Microbes' types and classes of VOCs are very wide, including fatty acid derivatives (hydrocarbons, alcohols, and ketones), aromatic compounds, nitrogen-containing compounds, and volatile sulfur compounds. Microbial volatile organic compounds (VOCs) can also be divided into several chemical classes: alkenes, alcohols, ketones, benzos, pyrazines, sulfides, acids, esters, and terpenes. This study aimed to design primers for genes encoding volatile synthesis in Ralstonia syzygii subsp. celebesensis, which causes blood disease in the banana plant. Some of the genes involved are adc (acetone synthesis), adhP (ethanol synthesis), ilvA, nirBD (ammonia synthesis), mdcA (propionic acid synthesis), cysI (hydrogen sulfide synthesis), and speBC (putrescine synthesis). Primers were designed and examined for specificity in silico using Primer3Plus, Geneious Prime, and BLAST programs. The numbers of nine pairs designed primers were successfully amplifying the related nine VOC genes of R. syzygii subsp. celebesensis for qPCR.

Sex determination based just on morphological traits such as plumage dichromatism, sexual size dimorphism, behavior, or vocalizations is really challenging because of the sexual monomorphism present in more than half of avian species. Currently, a lot of them can be tested through DNA-based procedures, but they do not fit all the avian species, such as Eudromia elegans. The aim of this study was to design a new molecular method suitable for routine sex determination for that species that is fast, simple, and cost- and time- effective. DNA was isolated from dry blood stain and/or chest feather samples of E. elegans species. We used two sets of sex-specific primers (ZF/ZR and WF/WR) to amplify the expected fragments localized on the highly conserved CHD1 gene to distinguish between sexes due to the W-specific DNA sequence present only in females. We confirmed the accuracy and consistency of the PCR-based method based on length differences to distinguish between the sexes of E. elegans, which amplified two fragments in females and one fragment in males.

Microbes produce various types of volatile organic compounds (VOCs) through metabolism, which can be used for diagnostic purposes. Microbes' types and classes of VOCs are very wide, including fatty acid derivatives (hydrocarbons, alcohols, and ketones), aromatic compounds, nitrogen-containing compounds, and volatile sulfur compounds. Microbial volatile organic compounds (VOCs) can also be divided into several chemical classes: alkenes, alcohols, ketones, benzos, pyrazines, sulfides, acids, esters, and terpenes. This study aimed to design primers for genes encoding volatile synthesis in Ralstonia syzygii subsp. celebesensis, which causes blood disease in the banana plant. Some of the genes involved are adc (acetone synthesis), adhP (ethanol synthesis), ilvA, nirBD (ammonia synthesis), mdcA (propionic acid synthesis), cysI (hydrogen sulfide synthesis), and speBC (putrescine synthesis). Primers were designed and examined for specificity in silico using Primer3Plus, Geneious Prime, and BLAST programs. The numbers of nine pairs designed primers were successfully amplifying the related nine VOC genes of R. syzygii subsp. celebesensis for qPCR. [ABSTRACT FROM AUTHOR]

Rapid and modular assembly of DNA parts is crucial to many synthetic biologists. This can be achieved through Golden Gate assembly, which often requires purchase and delivery of new primers for each part and assembly configuration. Here, we report on a small set of primers that can be used to amplify any DNA from the Registry of Standard Biological Parts for Golden Gate assembly. These primers bind to regions common to the backbone plasmid for these parts, but pair imperfectly and introduce type IIS restriction enzyme sites in a way that minimizes assembly scars. This approach makes redesign of assembly strategies faster and less expensive and can help expand access to synthetic biology to a wider group of scientists and students.

The aim of the study was to evaluate particular polymerase chain reaction primers targeting selected representative genes and the influence of a preincubation step in a selective broth on the sensitivity of group B Streptococcus (GBS) detection by nucleic acid amplification techniques (NAAT). Research samples were vaginal and rectal swabs collected in duplicate from 97 pregnant women. They were used for enrichment broth culture-based diagnostics, bacterial DNA isolation, and amplification, using primers based on species-specific 16S rRNA, atr and cfb genes. To assess the sensitivity of GBS detection, additional isolation of samples preincubated in Todd-Hewitt broth with colistin and nalidixic acid was performed and then subjected to amplification again. The introduction of the preincubation step increased the sensitivity of GBS detection by about 33–63%. Moreover, NAAT made it possible to identify GBS DNA in an additional six samples that were negative in culture. The highest number of true positive results compared to the culture was obtained with the atr gene primers, as compared to cfb and 16S rRNA primers. Isolation of bacterial DNA after preincubation in enrichment broth significantly increases the sensitivity of NAAT-based methods applied for the detection of GBS from vaginal and rectal swabs. In the case of the cfb gene, the use of an additional gene to ensure the appropriate results should be considered. [ABSTRACT FROM AUTHOR]

Rapid and modular assembly of DNA parts is crucial to many synthetic biologists. This can be achieved through Golden Gate assembly, which often requires purchase and delivery of new primers for each part and assembly configuration. Here, we report on a small set of primers that can be used to amplify any DNA from the Registry of Standard Biological Parts for Golden Gate assembly. These primers bind to regions common to the backbone plasmid for these parts, but pair imperfectly and introduce type IIS restriction enzyme sites in a way that minimizes assembly scars. This approach makes redesign of assembly strategies faster and less expensive and can help expand access to synthetic biology to a wider group of scientists and students. [ABSTRACT FROM AUTHOR]

We aimed to evaluate whether two commonly used PCR primers are effective to identify P. endodontalis and discriminate from other prevalent black-pigmented bacteria in apical periodontitis (AP). Endodontic canal samples from patients with asymptomatic AP (n = 20) were collected and cultured in anaerobiosis. Two primer sets to detect P. endodontalis were selected from the literature and first analyzed for their specificity in silico; and then tested on clinical isolates in vitro and finally, in apical exudates ex vivo. The identity of P. endodontalis was verified by PCR and Sanger sequencing with universal primers for bacterial V3–V6 regions 16S rDNA. Only one primer set showed specificity only for P. endodontalis clones in silico and also was specific for P. endodontalis in vitro and ex vivo. [ABSTRACT FROM AUTHOR]

Metabarcoding of microbial eukaryotes (collectively known as protists) has developed tremendously in the last decade, almost solely relying on the 18S rRNA gene. As microbial eukaryotes are extremely diverse, many primers and primer pairs have been developed. To cover a relevant and representative fraction of the protist community in a given study system, an informed primer choice is necessary, as no primer pair can target all protists equally well. As such, a smart primer choice is very difficult even for experts and there are very few online resources available to list existing primers. We built a database listing 285 primers and 83 unique primer pairs that have been used for eukaryotic 18S rRNA gene metabarcoding. In silico performance of primer pairs was tested against two sequence databases: PR2 version 4.12.0 for eukaryotes and a subset of silva version 132 for bacteria and archaea. We developed an R‐based web application enabling browsing of the database, visualization of the taxonomic distribution of the amplified sequences with the number of mismatches, and testing any user‐defined primer or primer set (https://app.pr2‐primers.org). Taxonomic specificity of primer pairs, amplicon size and location of mismatches can also be determined. We identified universal primer sets that matched the largest number of sequences and analysed the specificity of some primer sets designed to target certain groups. This tool enables guided primer choices that will help a wide range of researchers to include protists as part of their investigations. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate particular polymerase chain reaction primers targeting selected representative genes and the influence of a preincubation step in a selective broth on the sensitivity of group B Streptococcus (GBS) detection by nucleic acid amplification techniques (NAAT). Research samples were vaginal and rectal swabs collected in duplicate from 97 pregnant women. They were used for enrichment broth culture-based diagnostics, bacterial DNA isolation, and amplification, using primers based on species-specific 16S rRNA, atr and cfb genes. To assess the sensitivity of GBS detection, additional isolation of samples preincubated in Todd-Hewitt broth with colistin and nalidixic acid was performed and then subjected to amplification again. The introduction of the preincubation step increased the sensitivity of GBS detection by about 33–63%. Moreover, NAAT made it possible to identify GBS DNA in an additional six samples that were negative in culture. The highest number of true positive results compared to the culture was obtained with the atr gene primers, as compared to cfb and 16S rRNA primers. Isolation of bacterial DNA after preincubation in enrichment broth significantly increases the sensitivity of NAAT-based methods applied for the detection of GBS from vaginal and rectal swabs. In the case of the cfb gene, the use of an additional gene to ensure the appropriate results should be considered.

Introduction. In recent years the presence of reassortant rotavirus strains is increasingly mentioned in the world due to the application of the full-genome based classification system. Information on the circulation of such strains in the territory of Russia is limited. The aim of this work was the development of the approach for determination of genotypes of segments encoding VP6 (I) and NSP4 (E) to reveal reassortant strains. Material and Methods. Rotavirus-positive samples were studied by means of nucleotide sequencing and multiplex PCR. Phylogenetic analysis was conducted using the Bayesian approach. Results. Three alleles of the VP6 gene (I1-1, I2-IV, I2-VII) and seven alleles of the NSP4 gene (E1-I, E1-III, E2-VI, E2-VII, E2-X, E2-XII, E3) were detected on the base of nucleotide sequences of Nizhny Novgorod rotaviruses. Taking into account these results, the oligonucleotide primers specific to genotypes I1, I2, I3 and E1, E2, E3 were designed. Optimal conditions for multiplex PCR were chosen. The method was tested using the strains collected in Nizhny Novgorod in 2018. The diversity of I and E genotypes was determined and various combinations with G and P genotypes were identified. Discussion. G9-P[8]-I1-E1 rotaviruses were predominant (32.7 %) and G2-P[4]-I2-E2 rotaviruses were in second place (29.1 %). Strains with genotypes G4-P[8]-I1-E2, G3-P[8]-I2-E2 and G2-P[4]-I2-E1 were detected sporadically. They had genes of two rotavirus genogroups, so can be considered to be reassortant. Conclusion. The proposed approach is a useful tool for the characterization of rotaviruses in the conditions of the beginning of vaccination against rotavirus infection in Russia.

Many environmental and biomedical biomonitoring and detection studies aim to explore the presence of specific organisms or gene functionalities in microbiome samples. In such cases, when the study hypotheses can be answered with the exploration of a small number of genes, a targeted PCR-approach is appropriate. However, due to the complexity of environmental microbial communities, the design of specific primers is challenging and can lead to non-specific results. We designed PhyloPrimer, the first user-friendly platform to semi-automate the design of taxon-specific oligos (i.e., PCR primers) for a gene of interest. The main strength of PhyloPrimer is the ability to retrieve and align GenBank gene sequences matching the user’s input, and to explore their relationships through an online dynamic tree. PhyloPrimer then designs oligos specific to the gene sequences selected from the tree and uses the tree non-selected sequences to look for and maximize oligo differences between targeted and non-targeted sequences, therefore increasing oligo taxon-specificity (positive/negative consensus approach). Designed oligos are then checked for the presence of secondary structure with the nearest-neighbor (NN) calculation and the presence of off-target matches with in silico PCR tests, also processing oligos with degenerate bases. Whilst the main function of PhyloPrimer is the design of taxon-specific oligos (down to the species level), the software can also be used for designing oligos to target a gene without any taxonomic specificity, for designing oligos from preselected sequences and for checking predesigned oligos. We validated the pipeline on four commercially available microbial mock communities using PhyloPrimer to design genus- and species-specific primers for the detection of Streptococcus species in the mock communities. The software performed well on these mock microbial communities and can be found at https://www.cerealsdb.uk.net/cerealgenomics/phyloprimer.

Many environmental and biomedical biomonitoring and detection studies aim to explore the presence of specific organisms or gene functionalities in microbiome samples. In such cases, when the study hypotheses can be answered with the exploration of a small number of genes, a targeted PCR-approach is appropriate. However, due to the complexity of environmental microbial communities, the design of specific primers is challenging and can lead to non-specific results. We designed PhyloPrimer, the first user-friendly platform to semi-automate the design of taxon-specific oligos (i.e., PCR primers) for a gene of interest. The main strength of PhyloPrimer is the ability to retrieve and align GenBank gene sequences matching the user's input, and to explore their relationships through an online dynamic tree. PhyloPrimer then designs oligos specific to the gene sequences selected from the tree and uses the tree nonselected sequences to look for and maximize oligo differences between targeted and non-targeted sequences, therefore increasing oligo taxon-specificity (positive/negative consensus approach). Designed oligos are then checked for the presence of secondary structure with the nearest-neighbor (NN) calculation and the presence of off-target matches with in silico PCR tests, also processing oligos with degenerate bases. Whilst the main function of PhyloPrimer is the design of taxon-specific oligos (down to the species level), the software can also be used for designing oligos to target a gene without any taxonomic specificity, for designing oligos from preselected sequences and for checking predesigned oligos. We validated the pipeline on four commercially available microbial mock communities using PhyloPrimer to design genus- and speciesspecific primers for the detection of Streptococcus species in the mock communities. The software performed well on these mock microbial communities and can be found at https://www.cerealsdb.uk.net/cerealgenomics/phyloprimer. [ABSTRACT FROM AUTHOR]

Oxacillinases (OXA) have been mostly described in Enterobacteriaceae, Acinetobacter, and Pseudomonas species. Recent years have witnessed an increased prevalence of intrinsic and/or acquired β-lactamase-producing Acinetobacter in food-producing animals. This study was conducted to assess the prevalence of OXA among selected bacterial species and to characterize these enzymes by in silico analysis. Screening of OXA was performed by PCR amplification using specific pairs of oligonucleotides. Overall, 40 pairs of primers were designed, of which 6 were experimentally tested in vitro. Among 49 bacterial isolates examined, the presence of blaOXA-1-like genes was confirmed in 20 cases (41%; 19 times in Klebsiella pneumoniae and once in Enterobacter cloacae). No OXA were found in animal isolates. The study results confirmed the specificity of the designed oligonucleotide pairs. Furthermore, the designed primers were found to possess the ability to specifically detect 90.2% of all OXA. These facts suggest that the in silico and in vitro tested primers could be used for single or multiplex PCR to screen for the presence of OXA in various bacteria, as well as to monitor their spread. At the same time, the presence of conserved characteristic amino acids and motifs was confirmed by in silico analysis of sequences of representative members of OXA. [ABSTRACT FROM AUTHOR]

Aims: Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae's DNA detection based on polymerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence. Methods and Results: The specific target region, RLEP, of M. leprae's genome was selected based on comparative genomics. After confirming the specificity of this region, using blastn analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, whereas the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carried out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, whereas LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per millilitre, whereas LP1/LP2 was 1000 copies of bacterial genomes per millilitre. Conclusions: In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy. Significance and Impact of the Study: The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease. [ABSTRACT FROM AUTHOR]

This study was carried out in the biotechnology laboratories of the Seed Inspection and Certification Department - Ministry of Agriculture during 2019, to study the relation among molecular five inbred lines with hybrid crosses of maize. to estimate the genetic diversity and determine the degree of genetic similarity among them using the ISSR based on PCR technic with 6 primers. All primers produced 79 bands at a rate of 13.2 bands per primer. The number of amplified bands ranged from 10 to the primer UBC 835 and 16 in the UBC 897, with a molecular length ranged between 100 - 1700 bp. The total number and percentage of polymorphic bands was 71 and 90% respectively. The primers UBC 856 and UBC 866 the highest percentage of had polymorphic bands of 100%. While the UBC 835 showed the lowest (70%). The highest efficiency and specificity were 12.7% and 9.9% respectively in UBC835. Based on molecular marker data and genetic similarity using the UPGMA method in establishing the dendrogram, the study showed that genetic similarity between the inbred lines ranged from 0.105 to 0.40, the highest (0.40) between the two strains BK121 and Zm6 , While the lowest degree of similarity (0.105) was between the two strains BK121 and ART-817 . [ABSTRACT FROM AUTHOR]

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Identification has similarities and differences with other kinds of assessment activity: quality assessment, control management and certification. The final result of identification is verification of compliance or detection of falsification. Common features are tests for definition of actual values. This paper studies the design of universal primers for type identification of fruit raw material (strawberry, gooseberry, cherry, raspberry, banana, wild rose, kiwi). To further verify the specificity of primers, sequencing of fragments is produced, which are read by each from the primer pairs. For this purpose, 8 polymerase chain reactions (PCR-reactions) are initiated, one from each primer pair corresponding to one type of raw material. A single alignment matrix for each of the studied objects is created as a result. Re-verification of each matrix is conducted for the presence of read errors or other disputed single- nucleotide substitutions. It is stated that the alignment matrices of the nucleotide sequences of raspberry, strawberry (fragaria viridis), gooseberry, wild rose, cherry, banana and kiwi are aligned on all sides and the protruding "bases" do not disturb the future work of programmes for the primers design. Universal non-intersecting primers are chosen to identify the fruit raw material under studying. As a result of the use of various software packages and of the database GenBank NCBI, we managed to find a suitable DNA zone for each of the tested samples of fruit raw material at the level of generic differentiation for further development on its basis of the universal primers. It is zone 18S rDNA. All the found sequences have both the conservative part for planting a pair of primers, and the variable one for reliable identification of species or for phylogenetic analysis. As part of the study, all samples of fruit raw material have been identified

The objective of the study was to describe how much rat and swine primer developed from cytochrome b could detect rat and pork in processed beef products sold in North Maluku. The settings of the study were the traditional markets and supermarkets in several cities in North Maluku such as Ternate, Tidore Kepulauan, West Halmahera, North Halmahera, Central Halmahera, South Halmahera, East Halmahera, Sula Island and Morotai Island. The data collection lasted between May and June, 2015. The samples were analyzed in the Biotechnology Lab of Unkhair in July, 2015. To detect rat and swine DNA, the researchers used the PCR (Polymerase Chain Reaction) method with Top Taq master mix Kit kit (250) (Catalog no. 200403) Swine Primer: Forward: 5'CTA CAT AAG ATAT ATC CAC CAC A 3 'Reverse: 5' ACA TTG TGG GAT CTT CTA GGT 3 'Product size: 290 bp. Rat Primer: forward SIM (5'-GACCTCCCAGCTCCATCAAACATCTCATCTTGATGAAA-3'). Reverse (5'GAATGGGATTTT GTTGGAGTTT-3 '). Out of 41 samples, sample 3, 4 and 5 taken in Jailolo contained rat DNA (positive); the samples were amplified with 499 base pair length (bp). In addition, sample 2, 7, 8 and 10 from Ternate as well as sample 4 from Morotai Island was also found positive (containing rat DNA). In terms of swine DNA, all of the samples came back negative. The amplification showed that none of the meatball samples contained pork. No pig DNA was amplified in the gel.

Thermophilic microorganisms are of special interest for phylogenetics and research in prokaryotic evolution, since many of them belong to deep branches of the tree of life. For this reason a number of broadly used universal primer systems for the 16S rRNA gene eliminate certain groups of thermophilic prokaryotes from their detection spectra. In the present work, the known 16S rRNA gene primer systems were analyzed in order to determine their ability to reveal members of deep phylogenetic lineages containing thermophilic microorganisms. It was shown that application of most of the published primer systems could result in elimination of certain groups of thermophilic prokaryotes. In silico analysis of existing primer systems was used to select the primer system for the V3‒V4 region of the 16S rRNA gene, which minimized elimination of thermophilic prokaryotic groups. Comparison of the proposed system with the previously published ones was carried out using high-throughput sequencing. Statistical analysis of the sequencing results based on the Shannon and Chao1 indexes revealed high efficiency of the proposed system for analysis of microbial communities of Kamchatka hot springs. [ABSTRACT FROM AUTHOR]

Highlights • The taxon-specific primers were designed and evaluated in vitro and in silico for the phage identification. • The DNA markers identified in the present work can be useful for the taxon establishment of newly isolated phages. Abstract The phage taxonomy is primarily based on the morphology derived from Transmission Electron Microscopic (TEM) studies. TEM based characterization is authentic and accepted by scientific community. However, TEM based identification is expensive and time consuming. After the phage isolation, before analysis TEM, a DNA based rapid method could be introduced. The DNA based method could dramatically reduce the number of samples analyzed by TEM and thereby increase the speed and reduce the cost of identification. In the present work, four environmental phage isolates were identified based on TEM studies and genome size. The identification of these four phages was validated using DNA based method. The taxon-specific DNA markers were identified through multiple sequence alignments. The primers were designed at conserved genes (DNA polymerase or integrase) of 4 different phage taxa viz. family Ackermannviridae , genus Jerseyvirus , genus T4virus , and genus P22virus. These primers were evaluated using both in vitro and in silico approach for the amplification of the target taxons. Majority of the primer sets were found to amplify member species of the targeted taxa in vitro. In In silico analysis, six primer sets intended for identification of family Ackermannviridae showed positive amplification of ≥86.7% classified species. Further, the primers targeting the genus Jerseyviru s and T4viru s showed the amplification of 53.8% and ≥84.6% species, respectively. The present work is a case study performed to explore the possibility of use of taxon-specific primers for identification and taxonomic studies of newly isolated phages to supplement the TEM. [ABSTRACT FROM AUTHOR]

A polymerase chain reaction (PCR) assay for detecting avian leukosis/sarcoma viruses (ALSV) in chickens was developed, using primers we developed for specifically detecting subgroups A-E and J, ALSV infections. The performance of the assay was compared with that of published PCR methods, on DNA extracted from paraffinembedded tissues from chickens confirmed to be infected with ALSV subgroups A and J, by neutralization and ELISA tests. The results of some of our primer sets used in the assay agreed 100 percent with those of virus neutralization, and performed better than published PCR assays. The assay is presented as an alternative rapid, reliable and sensitive test for the detection of ALSV infections, that is equally as effective as virus neutralization tests which take days to complete, and may be a valuable diagnostic test in the eradication and control of ALSV infections in poultry flocks. [ABSTRACT FROM AUTHOR]

Mungbean Yellow Mosaic Virus is an important virus causing yellow mosaic disease in greengram. Yellow mosaic disease is one of the most destructive diseases of greengram. The economic losses due to this virus account up to 85 per cent in greengram which is spreading faster towards newer areas now-a-days and it is transmitted by the vector whitefly. Diagnosis of this viral disease at early stage is essential to prevent epidemics of the diseases and also viable management strategies could be designed. With an aim to device a rapid and accurate detection assay, Rolling Circle Amplification (RCA)-Polymerase Chain Reaction (PCR) technique was used for detection of the viral pathogen. RCA technique was employed to increase the viral template and PCR was conducted using a degenerate primer pair specific for MYMV coat protein region. The RCA product was subjected for restriction digestion with BamH1 and subjected to agarose gel electrophoresis which revealed the presence of ~2.7 kb DNA bands this shows that the involvement of begomovirus in the infected samples. Later RCA product subjected to PCR amplification using MYMV coat protein specific primers and 520 bp segment of MYMV was amplified. This method is simple and accurate and can help in early detection of viruses. [ABSTRACT FROM AUTHOR]

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created an exceptional situation in which numerous laboratories in Europe simultaneously implemented SARS-CoV-2 diagnostics. These laboratories reported in February 2020 that commercial primer and probe batches for SARS-CoV-2 detection were contaminated with synthetic control material, causing delays of regional testing roll-out in various countries. [ABSTRACT FROM AUTHOR]

A historical review of the advent and improvement of the methods for detecting multilocus DNA polymorphism that do not require preliminary knowledge of the individual gene and complete genome sequences of eukaryotes is presented. The first group of these methods includes approaches based on the use of primers with arbitrary sequence (random priming). Another group of methods to detect DNA polymorphism is based on the use of primers that consist of short repetitive sequences having anchor nucleotides at the 5'- or 3'-ends that position the annealing sites of these primers (microsatellite priming). Another approach for revealing polymorphism that does not require knowledge of the DNA sequence is based on cleavage of total DNA by a combination of restriction endonucleases (random cleavage) accompanied by PCR amplification. Considerable attention is paid to the opportunities of using these approaches to detect DNA polymorphism in the form of converting the obtained data to digital format and creation of integrative databases for all organisms, regardless of the methods used. [ABSTRACT FROM AUTHOR]

Using polymerase chain reaction 316 strains of E. coli (strains with normal enzymatic activity, strains with weak enzymatic activity and strains with hemolytic activity) were examined for the presence of pathogenicity genes. They were isolatedfrom healthy children and children with functional disorders of the gastro-intestinal tract. The studies have shown that strains of Escherichia coli have pathogenic potential, as evidenced by the presence of genetic pathogenicity markers in them.

Lactic acid bacteria play a very important role in food fermentation. For strain identification, characterization and protection, it is important to use highly discriminatory identification methods. In the present study, 37 strains isolated from vegetables and food products of Korea were identified using 16S rDNA gene sequencing and were found to be Leuconostoc mesenteroides . Further, molecular characterization of all the strains was performed using two RAPD primers, i.e. 239 and KAY3. All strains have shown RAPD profiles and the amplified products of the profiles ranged from 300 to 4000 bp with both the primers and only small differences in banding pattern were observed. Out of 37, maximum bands were observed in strain 11436 with 239 and strain 11260 with KAY3. With both the primers, the phylogenetic analysis revealed seven clades which could be further subdivided into groups. The dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA). The results showed that 16S rDNA sequencing and RAPD-PCR are suitable preliminary molecular tools for identification and characterization of bacteria. [ABSTRACT FROM AUTHOR]

Universal genus-specific primers for comparative analysis of two aligned 16S rRNA gene nucleotide sequences of lactic acid bacteria were constructed. The method to identify lactic acid bacteria and a comprehensive plan for their genus and species identification may be used to characterize isolated strains of the Lactobacillus genus bacteria and in quality control of foodstuffs enriched with Lactobacillus .

Nitrogen (N2) is an essential nutrient for plant growth; however, roots can only assimilate the reduced form of this element. Specific bacterial communities, known as nitrogen-fixing bacteria, can synthesize a nitrogenase enzyme complex, which converts N2 into plant-assimilable molecules, such as nitrates (NO3) and ammonium (NH4+); nifH gene encodes a highly conserved subunit of the bacterial nitrogenase complex. In the present work, bioinformatic analyses of primers targeting bacterial nifH genes were performed. Nitrogenase complex subunit encoded by the nifH gene was identified in Uniprot database, this protein sequence was aligned against nucleotide sequences using the BLAST (tblastn) tool. A total of 4,693 nucleotide sequences were obtained and aligned using MEGA11. Aligned sequences were subjected to an in-silico PCR assay using primer pairs identified in previous studies. These analyses allowed us to identify that primer pair (nifH2/nifH1) had the highest coverage percentage (80%). Results of this study will allow us to detect and quantify the presence of bacterial nifH genes in soil and root samples using PCR assays. [ABSTRACT FROM AUTHOR]

The genus Blastocystis is one of the most genetically diverse parasites. Blastocystis isolates from humans and animals have been classified into subtypes (STs) based on the phylogeny of the small subunit rRNA gene (SSU rDNA). Although human Blastocystis isolates are limited to STs 1–9, the identification of all 9 STs remains challenging due to the lack of specific primers for several STs. The sequencing of partial SSU rDNA is therefore essential for the identification of several STs. In this study, we developed 9 sets of PCR primers to detect each of the 9 kinds of ST in humans. When these ST-specific primer pairs were examined reference Blastocystis for the 9 STs, all 9 amplified only the target ST even in a DNA mixture of all 9 STs. The specificities of the 9 primer sets were tested against several intestinal parasites and fungi found in human stool samples. No amplification with these common human intestinal eukaryotes was observed using the primer pairs for 8 STs, while the ST5 primer set gave only faint bands with some parasites. Since genomic DNA levels of these parasites extracted from Blastocystis -positive cultures are expected to be markedly lower than the pure or highly concentrated DNA samples tested, the cross-amplifications with these organisms are unlikely to be detected when DNA samples are extracted from Blastocystis -positive cultures. The PCR conditions for all 9 primer sets were the same, hence a one-step analysis by PCR amplification, followed by electrophoresis has potential as a simple tool for the subtyping of human Blastocystis isolates. [ABSTRACT FROM AUTHOR]

Summary Tuberculous meningitis (TBM) is one of those most serious manifestations of extra-pulmonary tuberculosis and prompt diagnosis and treatment is required for better clinical outcome. It is difficult to diagnose due to lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the Multiplex polymerase chain reaction (PCR) technique, using primers directed against the insertion sequence IS6110 and MPB64 gene for the detection of Mycobacterium tuberculosis in Cerebrospinal fluid (CSF), for rapid diagnosis of TBM patients. 102 CSF samples were analyzed from patients suspected with TBM along with a control group of 10 patients having other neurological disorders. CSF sediments were analyzed individually for M. tuberculosis DNA by Multiplex PCR using two set of primers targeting insertion sequence IS6110 and gene MBp64, which is very specific for MTBC. Out of 37 patients diagnosed with TBM clinically, MPB64 PCR was positive in 22, IS6110 PCR was positive in 28, both PCR using Multiplex were positive in 34 and Microscopy was positive in one. Thus Sensitivity of MPB64 PCR, IS6110 PCR, Multiplex PCR and Microscopy were found to be 62.3%, 75.4%, 91.8% and 2.7% respectively. In non TBM group PCR was negative in all cases hence, the specificity was 100%. Multiplex PCR system using primers targeting IS6110 and MPB64 , for the detection of M. tuberculosis DNA in CSF samples, has high sensitivity than any one of them alone, and could be used for the early detection of TBM in CSF samples. [ABSTRACT FROM AUTHOR]

OBJETIVO: Determinar la frecuencia y distribución del virus del papiloma humano en los diferentes estadios que conforman la historia natural del cáncer cérvico uterino, y optimizar la detección mediante el uso de diferentes oligonucleótidos universales. MATERIAL Y MÉTODOS: Se trata de un estudio transversal, descriptivo, en el que las muestras fueron colectadas durante enero a diciembre de 1999. El procesamiento de las muestras y el análisis de los datos se realizaron en el Instituto Nacional de Cancerología en la Ciudad de México. Se hizo análisis comparativo con t de Student para valores continuos y con ji cuadrada para proporciones, y análisis de concordancia entre biopsia y exudado cervical con la prueba estadística de Kappa. Para la detección del virus se utilizó la técnica de la reacción en cadena de la polimerasa (PCR) con oligonucleótidos universales los cuales reconocen diferentes regiones del gen L1 (MY09/11; GP5/6; L1C1/2), y oligonucleótidos específicos para el VPH 16 y el VPH 18, así como secuenciación directa de los productos de la PCR. RESULTADOS: Se analizaron 154 muestras: 65 (42.2%) citologías normales, 45 (29.2%) lesiones de alto y bajo grado, y 44 (28.6%) de cáncer invasor. El VPH fue detectado en 95.5% de los casos de cáncer invasor, en 91.6% de lesiones de alto grado, en 66.7% de lesiones de bajo grado y en 23.1% de citologías normales, por la PCR con al menos uno de los juegos de oligonucleótidos utilizados. La detección fue más eficiente en las muestras obtenidas por biopsia que en los exudados cervicovaginales. El porcentaje total de detección del VPH con un juego de oligonucleótidos universales (37.6%) aumentó sustancialmente (60.4%) al combinarlo con otros dos juegos de oligonucleótidos universales. CONCLUSIONES: La presencia del VPH de alto riesgo es elevada inclusive en mujeres con epitelios cervicales con diagnóstico citológico normal. La detección del VPH mejora al utilizar distintos juegos de oligonucleótidos universales que reconocen la región L1 del VPH. Con una adecuada toma de muestra, el análisis para detección de ADN en exudado cérvico-vaginal es una buena alternativa de diagnóstico del VPH.OBJECTIVE: To determine the prevalence of human papillomavirus (HPV) infection at different stages of the natural history of cervical cancer. Also, to optimize its detection by means of different sets of general primers. MATERIAL AND METHODS: A descriptive, cross-sectional study was conducted between January and December 1999. Samples were processed and analyzed at the Instituto Nacional de Cancerología (National Cancerology Institute) in Mexico City. A comparative analysis was performed using Student's t for continuous values and the chi-squared test for proportions. A contingency analysis was made between biopsy and cervical exudates with the Kappa statistic. HPV detection was done by PCR with general primers which recognize different regions of the L1 gene (MY09/11; GP5/6; L1C1/2) and with HPV16- and HPV18- specific primers, as well as direct sequencing of PCR products. RESULTS: In total, 154 samples were analyzed: 65 (42.2%) of them showed normal cytology; 45 (29.2%) high and low grade lesions; and 44 (28.6%) invasive cervical cancer. HPV was detected in 95.5% of invasive cervical cancers, in 91.6% of high grade lesions, in 66.7% of low grade lesions, and in 23.1% of normal smears, by PCR with at least one set of oligonucleotide primers. HPV detection was more efficient in biopsy specimens than in cervical scrapes. The total percentage of HPV detection us ing only one set of universal oligonucleotides (37.6%)increased to 60.4% when the other two sets of universal oligonucleotides were used. CONCLUSIONS:The frequency of high risk HPV is high even in women with reported normal cytology. HPV detection improves when different sets of general primers directed to the L1 region are used. HPV DNA screening in cervical scrapes may be a good alternative HPV diagnostic tool when the samples are appropriately taken.