Polymerase Chain Reaction Products Research Articles

Avian Plasmodium spp. and Haemoproteus spp. parasites in mosquitoes in Germany

BackgroundAlthough haemosporidian parasites may cause considerable health and economic problems in aviaries, there is limited understanding of the vectors transmitting them. Mosquito-borne Plasmodium species are responsible for the deaths of numerous exotic (= immunologically naïve) birds in zoos every year, while native birds are adapted to the parasites and largely protected by an effective immune response.MethodsMosquitoes were collected in bird/animal parks, wetlands and private gardens in various regions of Germany from 2020 to 2022. Females were pooled with up to 10 specimens according to taxon, location and date. Extracted DNA was screened for avian Haemosporida-specific mitochondrial rDNA using real-time polymerase chain reaction (PCR). Positive samples were amplified by a Plasmodium/Haemoproteus-specific nested PCR targeting the partial cytochrome b gene, followed by sequencing of the PCR product for species identification. Sequences were checked against GenBank and MalAvi databases.ResultsPCR of 2633 pools with 8834 female mosquitoes signalled infection with Plasmodium in 46 pools and with Haemoproteus in one pool. Further amplification and sequencing demonstrated the occurrence of Haemoproteus majoris lineage PARUS1 (n = 1) as well as several Plasmodium species and lineages, including Plasmodium relictum SGS1 (n = 16) and GRW11 (n = 1), P. matutinum LINN1 (n = 13), P. vaughani SYAT05 (n = 10), P. circumflexum TURDUS01 (n = 3), P. cathemerium PADOM02 (n = 1) and Plasmodium sp. SYBOR02 (n = 1) and PLOPRI01 (n = 1). The infections were detected in Culex pipiens sensu lato (n = 40), Culiseta morsitans/fumipennis (n = 6) and Aedes cinereus/geminus (n = 1).ConclusionsAlthough the overall Plasmodium minimum infection rate (5.2) appears to be low, the results demonstrated not only the ongoing circulation of Plasmodium parasites in the German mosquito population, but also the occurrence of eight distinct Plasmodium lineages, with three of them (PADOM02, SYBOR02, PLOPRI01) being detected in Germany for the first time. This study highlights the importance of conducting mosquito-borne pathogen surveillance studies simultaneously targeting vectors and vertebrate hosts, as certain species may be detected more readily in their vectors than in their vertebrate hosts, and vice versa.Graphical

Polymorphisms in the promoter regions of RHD and RHCE genes in the Chinese Han population.

The Rh blood group system is the most polymorphic human blood group system. Previous studies have investigated variants in the RHD and RHCE promoter. The relevance of these variants to the Chinese Han population is further clarified in this study. In total, 317 donors (223 Rh D-positive [D+], including 20 Del and 94 Rh D-negative [D-]) were randomly selected. The promoter regions and exon 1 of RHD and RHCE were amplified through polymerase chain reaction (PCR)whose products were directly sequenced using forward and reverse primers. Expected PCR products of the RHD promoter and exon 1 were amplified in 223 D+ individuals, including 20 Del individuals, and were absent in 81 of 94 D- individuals. Expected PCR products of RHCE were observed in all donors. Two single nucleotide variants (SNVs) were observed in the RHD promoter region. Moreover, 11 SNVs were observed in the promoter and exon 1 of RHCE. rs4649082, rs2375313, rs2281179, rs2072933, rs2072932, rs2072931 and rs586178 with strong linkage disequilibria were significantly different between the D+ and D- groups. [A;C] was the most common haplotype in the RHD promoter (NC_000001.11:g.[-1033A>G;-831C>T]). [G;T;T;A;T;A;C;G;A;C;G] was the most predominant haplotype in both total and D- groups. In D+ individuals, [A;C;T;G;C;G;C;G;C;C;C] was the most frequent haplotype in the RHCE promoter (NC_000001.11:g.[-1080A>G;-958C>T;-390T>C;-378G>A;-369C>T;-296G>A;-144C>G;-132G>A;-122C>A;28C>T;48C>G]). We speculate that the SNVs/haplotypes found in this article cannot significantly affect gene expression. The present study findings should help elucidate the molecular basis of the polymorphic expression of RHD and RHCE promoter regions.

Widespread occurrence of Tilapia parvovirus in farmed Nile tilapia Oreochromis niloticus from India.

Tilapia parvovirus (TiPV) has been associated with heavy mortalities in tilapia as a single infection or in co-infection with Tilapia lake virus (TiLV). In this study, TiPV was detected in farmed Nile tilapia, Oreochromis niloticus, from two geographical regions of India, Maharashtra and Uttar Pradesh. TiPV-specific polymerase chain reaction (PCR) reported earlier was used in the screening. Tilapia collected from Maharashtra showed characteristic clinical signs, and TiPV was detected along with TiLV and/or Aeromonas spp. However, fish from Uttar Pradesh were apparently healthy and only TiPV could be detected in these samples. A high prevalence of TiPV was recorded from both the geographical locations, Maharashtra and Uttar Pradesh (59.6% and 95.0% respectively). The virus could be detected in tissues such as the spleen, liver, kidney, brain and mucus. The spleen appeared to be the best tissue for detecting TiPV in apparently healthy tilapia. The presence of TiPV was further confirmed through sequencing the PCR products, isolation of the virus in the cell line and electron microscopy. Sequences of the NS1 gene of the two TiPV isolates showed similarity to the earlier reported TiPV isolates. The virus could be successfully propagated in O. niloticus Liver (OnL) cell line, and cytopathic effect was observed as early as 3 days post-infection. Furthermore, the presence of non-enveloped icosahedral to round virus particles measuring about 26-35 nm could be demonstrated in the cytoplasm and nucleus of infected OnL cells in transmission electron microscopy. With this confirmation of the presence of the virus, India is the third country to report TiPV after China and Thailand. The detection of TiPV in co-infection cases with TiLV and in apparently healthy Nile tilapia suggests its wide distribution and potential synergistic effect in co-infection cases. Therefore, this emerging virus needs holistic attention to understand its virulence, host-specificity and epidemiological risk factors.

Characterization of type I L-asparaginase encoding gene from the thermohalophilic bacterium Bacillus subtilis CAT3.4 from Wawolesea Hot Spring, Southeast Sulawesi, Indonesia

Abstract. Muzuni, Ilham M, Ardiansyah, Suriana, Putri Y, Kartina. 2023. Characterization of type I L-asparaginase encoding gene from the thermohalophilic bacterium Bacillus subtilis CAT3.4 from Wawolesea Hot Spring, Southeast Sulawesi, Indonesia. Biodiversitas 24: 5167-5178. This study aims to determine the molecular characteristics of the gene encoding type 1 L-asparaginase from CAT3.4 thermohalophilic bacterial isolates from the hot springs of Wawolesea, North Konawe, Southeast Sulawesi. This enzyme can be used as a cancer therapy agent and prevents the formation of acrylamide in food products. It is a kind of exploratory research. The characterization was done by amplifying the ansA gene sequence encoding L-asparaginase from CAT3.4 isolate by polymerase chain reactions (PCR) technique using primers AsnBac1-F1 (5'-ACGCGATATTTCTTTTGGCCGG-3') and AsnBac1-R1 (5'-CAGTGAAGAGGTGCATGGTATG-3'). The amplified PCR product was used as a template for sequencing by the Sanger method. The amino acid coding regions (CDS) obtained were bioinformatically characterized using the NCBI website for BLASTn analysis, restriction site and hydrophobicity profiles were analyzed using the BioEdit program, phylogenetic tree was analyzed using the MEGA X program, and type 1 L-asparaginase amino acid sequence was analyzed using the Expasy translate program. The characterization results showed that the target gene has a high similarity to the ansA gene sequence of 20 Bacillus subtilis strains, i.e., 99-100%, is closely related to the ansA gene of Bacillus subtilis strain SRCM103629 and Bacillus subtilis strain GOT9, can be identified using restriction enzymes MluI and BstI to differentiate the species of organisms they produce, has CDS encoding 329 amino acids with a dominant composition of polar amino acids (54.1%) and has a hydrophobicity profile of amino acids dominated by hydrophilic regions. All these characteristics have confirmed that the characterized gene is the ansA gene encoding type 1 L-asparaginase from Bacillus subtilis. The finding of the ansA gene from Bacillus subtilis in a thermohalophilic region in Southeast Sulawesi, Indonesia is a novelty of the research.

An idea to explore: Determination of single nucleotide polymorphisms in alcohol metabolism-related genes using PCR-based assays to understand the link between an individual's genotype and phenotype.

Here, we propose a laboratory exercise to quickly determine single nucleotide polymorphisms (SNPs) in human alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes involved in alcohol metabolism. In this exercise, two different genotyping methods based on polymerase chain reaction (PCR), namely allele-specific (AS) PCR and a PCR-restriction fragment polymorphism (RFLP) analysis, can be performed under the same PCR program (2-step × 35 cycles, 35 min total) in parallel using a hair root lysate as a template. In AS-PCR, the target regions of the G- or A-alleles of both genes are allele-specifically amplified in a single PCR tube. In the PCR-RFLP analysis, the two genes are amplified simultaneously in a single tube, and then a portion of the PCR product is double-digested with restriction enzymes MslI and Eam1104I for 5 min. The resulting reaction products of each method are electrophoresed side by side, and the genotypes are determined from the DNA band patterns. With the optimized protocol, the whole process from template preparation to genotyping can be completed in about 75 min. During PCR, students also perform an ethanol patch test to estimate their ability to metabolize alcohol. This series of experiments can help students learn the principles and applications of PCR/SNP analyses. By comparing the genotypes revealed by PCR and the phenotypes revealed by the patch tests, students can gain a better understanding of the clinical value of genetic testing.

Genetic analysis challenges the presence of Ixodes inopinatus in Central Europe: development of a multiplex PCR to distinguish I. inopinatus from I. ricinus

BackgroundIxodes ricinus is an important vector of several pathogens, primarily in Europe. Recently, Ixodes inopinatus was described from Spain, Portugal, and North Africa and then reported from several European countries. In this study, a multiplex polymerase chain reaction (PCR) was developed to distinguish I. ricinus from I. inopinatus and used in the surveillance of I. inopinatus in Algeria (ALG) and three regions in the Czech Republic (CZ).MethodsA multiplex PCR on TROSPA and sequencing of several mitochondrial (16S rDNA, COI) and nuclear markers (TROSPA, ITS2, calreticulin) were used to differentiate these two species and for a subsequent phylogenetic analysis.ResultsSequencing of TROSPA, COI, and ITS2 separated these two species into two subclades, while 16S rDNA and calreticulin could not distinguish I. ricinus from I. inopinatus. Interestingly, 23 nucleotide positions in the TROSPA gene had consistently double peaks in a subset of ticks from CZ. Cloning of these PCR products led to a clear separation of I. ricinus and I. inopinatus indicating hybridization and introgression between these two tick taxa. Based on a multiplex PCR of TROSPA and analysis of sequences of TROSPA, COI, and ITS2, the majority of ticks in CZ were I. ricinus, no I. inopinatus ticks were found, and 10 specimens showed signs of hybridization. In contrast, most ticks in ALG were I. inopinatus, four ticks were I. ricinus, and no signs of hybridization and introgression were detected.ConclusionsWe developed a multiplex PCR method based on the TROSPA gene to differentiate I. ricinus and I. inopinatus. We demonstrate the lack of evidence for the presence of I. inopinatus in Central Europe and propose that previous studies be re-examined. Mitochondrial markers are not suitable for distinguishing I. inopinatus from I. ricinus. Furthermore, our data indicate that I. inopinatus and I. ricinus can hybridize, and the hybrids can survive in Europe.Graphical abstract

Genetic Diversity within Scorpio Genus (Scorpion: Scorpionidae) from Iran: Preliminary Evidence based on 16srRNA Sequence.

The yellow digger scorpion, Scorpio maurus, is a medically important scorpion for which little is known about its genetic diversity. Polymerase chain reaction products of 16srRNA gene fragments were generated from scorpion specimens named SmKh1 and SmKh2. These sequences showed high similarity with the only partial sequence of S. maurus isolate SCA1 large subunit ribosomal RNA gene available in the Genbank database. The drawing of the phylogeny tree showed two clusters, A and B. The two specimens (SmKh1 and SmKh2), which are placed in sub-cluster A2, were provided from Behbahan, Iran, and they have the closest relationship with the only sequence of S. maurus (MW281771), which is also collected from Behbahan. It is noteworthy that the two sequences obtained from S. maurus scorpions recorded from Miandoab (MK170444) and Mahabad (KU705354), which are in sub-cluster A1, are more similar to the scorpions isolated from the Mediterranean basin than those collected from Behbahan. This issue is probably due to the fact that patterns of genetic diversity are a reflection of variation in gene flow, which is also influenced by factors such as territorial barriers and geographical distances. We conclude that the scorpions of this study accompanied by similar scorpions in the Mediterranean basin, belong to the same species despite the insignificant differences.

Identification of Candida Isolates from Cancer Patients Using Multiplex PCR

Aim of this study was to identify the Candida isolates from Cancer Hospital Maharagama, Sri Lanka by using Multiplex PCR (polymerase chain reaction) method. In this study, Multiplex PCR was used to identify Candida isolates from cancer patients at Apeksha Hospital, Sri Lanka. In multiplex PCR, 18 specific primers (nine primer pairs) were separated into three groups for multiplex PCR (PS I, PS II and PS III). Different sized PCR products corresponding to each Candida species were produced by PCR using the primer mixes. Among 52 Candida isolates, 49 were identified using the multiplex PCR procedure. Candida tropicalis was found to be the most prevalent species (38%), followed by Candida parapsilosis (31%), Candida albicans (13%), Candida glabrata (8%) and Candida krusei (4%). The study concludes that multiplex PCR is a better approach for the identification of Candida species and recommends the use of it for clinical and diagnostic purposes and for research purposes.

Meta-genomic Analysis of Parasites of Hyalomma Ticks of Cattle

Introduction: Ticks are the obligate blood-feeding parasites responsible for transmitting various pathogens to domestic animals and humans. Ticks and tick-borne diseases are a continuous threat to livestock development in tropical and subtropical areas of the globe. To the best of our knowledge, this was the first study on the microbiome population of Hyalomma spp. infesting livestock in Pakistan. Methods: Ticks were collected from hemo-parasite-positive cattle and were identified using standard entomological keys. Then, selected ticks were kept in the biochemical oxygen demand (BOD) incubator for egg laying and hatching at optimum temperature and humidity. A total of 16 (saliva and gut) samples were amplified through the reverse line blotting (RLB) primer of which 9 out of 16 were positive. Out of 9 positive samples, 4 samples of saliva and 5 samples of gut showed a polymerase chain reaction (PCR) product of 390 bp on 1.5% agarose gel stained with cyber green safe gel dye. Similarly, all 16 samples were amplified through the PCR by applying primers specific to the genus Anaplasma. Out of 16 amplified samples (8 saliva and 8 gut), 9 samples exhibited a positive amplification of 345 bp on 1.5% Agarose gel. After the identification of the genus, all 16 samples were subjected to species identification through PCR by applying primers specific to Theileria annulata, Babesia bovis, and Babesia bigemina. Results: The results indicated that 9 out of 16 samples are positive for Theileria, showing a PCR product of 460 bp on Agarose gel, while none of the single samples was positive for B. bovis and B. bigemina, respectively. Overall, 16 samples were subjected to metagenomics analysis within the saliva and gut of Hyalomma ticks, and consequently, Anaplasma and T. annulata were identified. Hence, it was found that the Hyalomma tick reported in Pakistan is responsible for the transmission of T. annulata and Anaplasma spp., and both parasites are responsible for causing theileriosis and anaplasmosis in cattle. Conclusion: It can be concluded that Hyalomma anatolicum transmits both T. annulata and Anaplasma species but not Babesia species in cattle. Thus, H. anatolicum ticks are potent vectors for T. annulata and Anaplasma species.

The evaluation of the possibility of Li-Fraumeni syndrome in cancer patients in East Azarbaijan Province of Iran

Introduction: In 1969, Li-Fraumeni syndrome (LFS), which is a rare cancer predisposition syndrome, was reported for the first time. The main problem in LFS is the mutation in the TP53 gene, which is a crucial tumor suppressor gene in the cell cycle. A hereditary syndrome is inherited in an autosomal dominant pattern. There is a significant correlation between this syndrome and various cancers such as sarcoma, breast cancer, brain tumors, and different other types of malignancies. This study aimed to identify the possibility of LFS in cancer patients in the East Azarbaijan, Iran. Methods: In this experimental study, 45 children with cancer in the Northwest of Iran were investigated for LFS. DNA was extracted from the whole blood cells using the salting-out method. The region within the exons 5-8 of the TP53 gene has been replicated via Polymerase Chain Reaction (PCR) method. The PCR products were sent for Sanger sequencing, and finally, the data were analyzed by Chromas software. Results: In the studied probands, in 12 (26.67%) cases, polymorphisms in Exon 6 and Introns 6 and Intron 7 were identified, and no mutation was observed in exons 5-8 of the TP53 gene. Conclusion: Our results show that there were no mutations in exons 5-8 of the TP53 gene as an indication of LFS possibility in these families. Further studies are needed to be done in a bigger population, and Next-Generation Sequencing (NGS) needs to be done to evaluate the whole genome of these patients to complete our data.

Borrelia persica infection in wild carnivores in Israel: molecular characterization and new potential reservoirs

BackgroundBorrelia persica causes tick-borne relapsing fever in Israel, the eastern Mediterranean basin, and Asia. Relapsing fever is associated with severe illness and potentially death in humans and animals. Since B. persica infection has rarely been described in wild animals, the aim of this study was to evaluate the prevalence of infection with B. persica in wild carnivores in Israel.MethodsSpleen and blood clot samples from wild carnivores, which underwent necropsy, were tested for the presence of Borrelia DNA by real-time polymerase chain reaction (PCR). PCR products were sequenced, and the spirochete loads were quantified using a specific quantitative PCR (qPCR).ResultsA total of 140 samples from 74 wild carnivores were analyzed for the presence of Borrelia DNA. Six out of the 74 (8.1%) animals were found positive for B. persica by PCR and sequencing of the flagellin B gene, of which 4/74 (5.4%) were also positive by PCR for the glycerophosphodiester phosphodiesterase (glpQ) gene. Positive samples were obtained from three European badgers, and one striped hyena, golden jackal, and red fox each. All B. persica-positive animals were young males (P < 0.0001). Quantifiable results were obtained from 3/5 spleen and 4/5 blood samples. The spirochete loads in the blood were significantly higher than those found in the spleen (P = 0.034).ConclusionsThe prevalence of B. persica infection found in wild carnivores brought for necropsy was unexpectedly high, suggesting that this infection is widespread in some wild animal species in Israel. This is the first report of B. persica infection in the European badger and striped hyena. These carnivores have a wide geographical range of activity, and the results of this survey raise the possibility that they may serve as reservoir hosts for B. persica.Graphical

Molecular evidence of Rickettsia canadensis in ticks, Hebei, China

China was affected severely by tick-borne rickettsiosis, and more than 10 Rickettsia species pathogenic to humans have been identified. In recent years, several Rickettsia members, with unknown pathogenicity, firstly identified abroad have been found in China. In this study, parasitic and questing ticks were recovered from two sampling sites in Hebei, China. Specific primers targeting outer membrane protein B (ompB) gene were designed to test the presence of Rickettsia canadensis by nested Polymerase Chain Reaction (PCR). As a result, a total of 428 ticks, including 232 ticks (including 230 Haemaphysalis longicornis and two H. japonica) from Laiyuan County and 196 (H. longicornis) from Luanping County, were collected. Sequencing of PCR products with the expected size and subsequently BLAST showed that 38H. longicornis ticks tested positive for R. canadensis, with an overall positive rate of 8.8%. In addition, 800-bp ompB gene and nearly complete citrate synthase (gltA) gene were recovered from six randomly selected positive samples to better understand their genetic characteristics. Nucleotide identity and phylogenetic analyses showed that R. canadensis presented geographical clustering with evidence that variants identified in the current study presented closer genetic relationship with others identified in Asian than those found in North America. In addition, epidemiological data suggested that H. longicornis may be the competent vector, and more attention should be paid to R. canadensis due to its zoonotic potential. In sum, R. canadensis was confirmed to be present in Hebei Province, China, and its surveillance in ticks should be strengthened due to potential pathogenicity, higher positive rate in ticks and wide distribution of possible vector tick species.

Polymerase incorporation of fluorescein or rhodamine modified 2′-deoxyuridine-5′-triphosphates into double-stranded DNA for direct electrochemical detection

The 2′-deoxyuridine-5′-triphosphates modified with fluorescein (dUTP-Fl) or rhodamine (dUTP-Rh) were tested as bearers of electroactive labels and as proper substrates for polymerases used in polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) with the aim of electrochemical detection of double-stranded DNA (dsDNA) amplification products. For this purpose, electrochemical behavior of free fluorescein and rhodamine as well as the modified nucleotides, dUTP-Fl and dUTP-Rh, was studied by cyclic (CV) and square wave (SWV) voltammetry on carbon screen printed electrodes. Both free fluorescein and dUTP-Fl underwent a two-step oxidation at the peak potentials (Ep) of 0.6–0.7 V and 0.8–0.9 V (phosphate buffer, pH 7.4). The reduction peaks of fluorescein and dUTP-Fl were registered between –0.9 V and −1 V, but they did not depend on concentration. The free rhodamine and dUTP-Rh have demonstrated the well-defined oxidation peaks at 0.8–0.9 V. In addition, the distinct reduction peaks at Ep between –0.8 V and −0.9 V were registered for both rhodamine and dUTP-Rh. The dUTP-Fl and dUTP-Rh were further tested as substrates to incorporate an electroactive label into 210 or 206 base pair long dsDNA amplicons generated either by PCR or RPA. Among two dUTP derivatives tested, dUTP-Fl revealed significantly better compatibility with PCR and RPA, producing the full-size amplicons at 50–90% substitution of dTTP in the reaction mixture. In the PCR, the best compromise between amplicon output and labeling was achieved at the dUTP-Fl : dTTP and dUTP-Rh : dTTP molar ratios of 70% : 30% and 20% : 80% in the PCR mixture, respectively, allowing the direct electrochemical detection of amplicons at micromolar concentrations. Alongside with fluorescence DNA assays, the fluorescein and rhodamine modified dUTP appear as promising electroactive labels to develop direct electrochemical DNA assays for detecting PCR and RPA products.

Molecular Detection of Borrelia spp. in Ticks of Sheep and Goats by Nested PCR Method in West Azerbaijan Province, Iran.

Background: Ticks are an important vector among arthropods associated with serious medical and veterinary problems. In this research, we investigate Borrelia species in ticks isolated from the surface of livestock (sheep and goat) in different regions of West Azerbaijan province. Materials and Methods: Polymerase chain reaction (PCR) was performed using specific primers targeting Borrelia spp. genes. Borrelia spp. was identified through PCR. The positive PCR products were sent to Pishgam Company for sequencing. Sequenced data were analyzed, and phylogenetic analysis was performed using maximum likelihood method in MEGA V.10. Results: The detection rate of Borrelia spp. for 16srRNA gene 69 (n = 542; 12.7%; 95%Cl: 10.1%-15.8%), 42 (n = 542; 7.7%; 95%Cl: 5.78%-10.1%) positive on 5S-23SrRNA gene and ospA gene 4 (n = 542; 0.74%; 95%Cl: 0.29%-1.88%). Conclusion: These results are the first report in Iran to identify Borrelia spp. These results of the study showed that the Borrelia spp. in hard ticks detected by PCR and it was negative to soft ticks. it is important in public health implications at the studied areas.

Morphological and Molecular Characterisation of Nineteen (19) Tomato Cultivars from Northern Nigeria

Tomato is an autogamous vegetable crop whose genetic variability has been reduced by selection and breeding processes. Characterisation of tomato germplasm, therefore, aids the documentation of the genetic variability existing in a population. To morphologically characterise tomato varieties from Northern Nigeria, and molecularly characterise the varieties using (Simple Sequence Repeat (SSR) markers, nineteen (19) tomato varieties were collected from major markets in seven states in Northern Nigeria using a purposive sampling method. Tomato seeds were extracted, prepared, sowed in screen houses and morphologically characterised using cross-sectional shapes, core, pericarp, matured fruit colour, overall fruit shape, size and firmness. Leaves of tomato seedlings four weeks after sowing were pooled and used for genomic DNA extraction. The polymerase chain reaction (PCR) was performed and PCR products were visualized through electrophoresis. SSR fragment analysis was scored as the presence or absence of a specific fragment size from the Electropherograms.&#x0D; Results showed that 44% of the colour of tomato core was white, 17% were red and pink, 5% were orange and yellow and 11% were variants of other colours, showing wide variations in the qualitative traits. Similarities among the nineteen tomato samples ranged from 14 (Dan India with Derica sweet, Kaduna Tangino, Nassarawa Roma 1 and 2, Niger Roma2, UTC01 and UTC 02) to 86% (between Mai mara and Derica small). SSR gave three unique SSR bands that identified and characterised the cultivars. &#x0D; The result of this study revealed genetic diversity within a group of tomato genotypes of different origins cultivated in Northern Nigeria using molecular (SSR) analysis. This confirmed the efficacy of the SSR markers as highly variable markers that detect the codominant single locus and are suitable to distinguish between the genetically related cultivars.

Comparison of 3 DNA extraction methods for extracting DNA from an adult Culex quinquefasciatus (Diptera: Culicidae).

The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from an adult Culex quinquefasciatus by focusing on the quantity and purity of DNA, cost, and time required. Ten mosquitoes were individually used for DNA extraction by each method. Based on the results obtained, DNA was extracted from each method using specific primers, resulting in a polymerase chain reaction (PCR) product with a length of 274 bp. The DNA quantity extracted using the DNAzol Direct (179.08 ± 3.77 ng/µl) differs significantly from that of the commercial kit (115.98 ± 4.57 ng/µl) and a microwave-based method (119.26 ± 3.06 ng/µl). The absorbance ratio of DNA extracted using the PureLink Genomic DNA kit, the DNAzol Direct, and the microwave-based methods was 1.92 ± 0.02, 1.79 ± 0.01, and 1.87 ± 0.01, respectively. Among the 3 methods evaluated, the microwave-based method is simpler, less expensive, and more time efficient. This is the first evaluation of the microwave-based method for extracting DNA from an adult mosquito. This study provides a useful guide for alternative DNA extraction methods for PCR-based assays, especially in low-resource settings.

Prevalence and molecular characterization of Giardia intestinalis isolated from children and calves in Babylon province, Iraq.

Giardia intestinalis is one of the most prevalent intestinal parasites in humans and animals, and children in close contact with livestock are particularly at risk of infection. This study aimed to detect assemblages of G. intestinalis and determine the origin of zoonotic transmission of Giardia in children and calves in different parts of Babylon province, Iraq. One hundred stool samples from children (68 boys and 32 girls) and 100 fecal samples from calves (46 males and 54 females) of different ages were randomly collected. Molecular techniques were used to estimate the prevalence of G. intestinalis in children and calves. A nested polymerase chain reaction (PCR) was performed by targeting the triose phosphate isomerase gene in the samples to detect G. intestinalis assemblages. The overall rates of infection with G. intestinalis in children and calves were 21% and 34%, respectively, using the conventional microscopic method. The results illustrated that 61.90% (13/21) and 38.09% (8/21) of positive samples from children were allocated to assemblages A and B, respectively (p > 0.05). In calves, assemblages A and B were detecte in 82.35% (28/34) and 17.64% (6/34) of positive samples from calves, respectively (p ≤ 0.001). Ten PCR products were sequenced and submitted to the GenBank database. Phylogenetic analysis detected five human sequences each belonging to G. intestinalis assemblages A (OM850335-OM850339) and B (OM850340-OM850344). Similarly, five calf sequences each belonged to G. intestinalis assemblages A (ON75756-ON757660) and B (ON757661-ON757665). The detection of large numbers of G. intestinalis assemblage A in both humans and cattle indicated that cattle could be a main source of zoonotic G. intestinalis infection in children in Babylon province, Iraq.

Molecular identification of phlebotomine sand flies and the harbored Leishmania spp. in Sokoto State, Nigeria.

Female sand flies are hematophagous, feeding on animals and in the process serve as vectors for Leishmania, the parasites that cause leishmaniasis in humans. Leishmaniasis are a group of parasitic neglected tropical diseases in 98 countries including Nigeria and kills ~60,000 people/year. In Nigeria, Sokoto State is endemic to leishmaniasis but there is a knowledge gap on the identity of the prevalent sand flies and the Leishmania species they transmit. Hence, this cross-sectional study was designed to take inventory of the species of sand flies in Sokoto using genetic methods. 1,260 (310 females) sand flies were collected from three Local Government Areas (L.G.A) of Sokoto State- Wamakko, Sokoto South and Kware. Genomic DNA was extracted from each fly and DNA amplification by polymerase chain reaction (PCR) was carried out on the DNA samples using primers targeting the arthropods mitochondrial cytochrome oxidase subunit 1 (mt-coI) gene, and nested PCR with primers targeting the gene for Leishmania internal transcribed spacer-1 (its-1) of ribosomal RNA its-1rRNA. The PCR products were sequenced. Gene sequence analysis revealed five species of sand flies belonging to the old-world genera namely Phlebotomus and Sergentomyia. The identified species were P. papatasi (6.45%), S. adleri (6.45%), S. affinis (9.7%), S. distincta (9.7%), S. schwetzi (67.7%). Within the sampling period, sand flies were most abundant in the rainy months of August (104/33.5%) and September (116/37.4%) with all the five identified species occurring. Sequence analysis of its-1 gene identified Leishmania infantum in two sand flies (2/310)- P. papatasi (from Sokoto South) and S. affinis (from Wamakko). BLAST search in NCBI and phylogenetic analysis revealed that the sand fly species are related to the species reported in different parts of Africa, while the L. infantum is identical to strain reported in Brazil (KY379083.1). Phlebotomus papatasi and four species belonging to the genus Sergentomyia are the most prevalent sand flies in Sokoto State, Nigeria and they harbor L. infantum solely. The results shed light on why visceral leishmaniasis is the most predominant form of the disease. Therefore, we recommend that adequate care for dogs must be instituted as dogs are the major animal reservoir for L. infantum.

Identification of animal species by nucleic acid chromatography of hair samples and investigation of its applicability to human biological samples

GeneFields®-Hair is a simple analysis kit that uses nucleic acid chromatography and polymerase chain reaction (PCR) to identify animal species of hair-like food contaminants. In this study, we evaluated GeneFields®-Hair as a simple and rapid method for identifying animal species from hair-like materials collected in forensic science, such as at crime scenes. The use of this kit with other human biological materials (whole blood, head dandruff, nails, saliva, oral mucosa, sebum, and urine) was also investigated. Animal body hair samples were pretreated by grinding in a buffer solution, centrifuged, and the supernatant was used for PCR. Nucleic acid chromatography of the PCR products allowed the identification of the animal species by the presence or absence of coloration on the decision line. For human biological materials, nucleic acid chromatography was performed after the appropriate pretreatment like body hair material. The determination of some animal species was difficult, even if they had a dedicated DNA Strip determination line. Furthermore, animals from the same family but different genera were sometimes detected on the same determination line. All the human biological samples were correctly identified. Smartphone photographs of the coloration of the judgment line were processed using the ImageJ software for quantitative determination.

First Report of Rhizopus stolonifer Causing Premature Soft Rot of Jackfruit in Bangladesh.

The jackfruit tree (Artocarpus heterophyllus) is native to South and South-east Asia including Bangladesh. It is a commercially important tropical tree species that produces fruit, food, fodder, and high-quality wood (Gupta et al. 2022). During surveys in February 2022, soft rot on immature fruit at approximately 70% incidence was observed in several plantations and homesteads in the Sylhet district of Bangladesh. Infected fruit had black patches surrounded by wide bands of white, powdery masses. The patches enlarged with fruit maturation, and in some cases, covered the entire fruit. Symptomatic fruit were collected, surface sterilized with 70% ethanol for 1 min, and washed 3 times with sterile distilled water. Fen air-dried, and small pieces from the margins of lesions were transferred to potato dextrose agar (PDA). The plates were incubated at 25°C in the dark. Two-day-old colonies had diffuse, gray cottony mycelia that were hyaline and aseptate under the microscope. Sporangiophores measuring 0.6-2.5mm in length and 18 to 23μm in diameter had rhizoids and stolons at their bases. Sporangia were almost spherical and were 125μm (±65μm, n=50) in diameter. Sporangiospores were ellipsoid to ovoid and measured 3.5 to 9.32μm × 2.82 to 5.86μm (x̄= 5.86×4.1μm, n=50). Based on these morphological features, the isolates were identified preliminarily as Rhizopus stolonifer (García-Estrada et al. 2019; Lin et al. 2017). To identify the pathogen molecularly, genomic DNA was extracted using the FavorPrep Fungi/Yeast Genomic DNA extraction Mini Kit (Taiwan). Polymerase chain reaction (PCR) amplification of ITS1-5.8S-ITS2 rDNA was done using primers ITS4 and ITS5 (White et al. 1990) following the procedure of Khan and Bhadauria (2019). The PCR product was then sequenced by Macrogen, Korea. A BLAST search in GenBank revealed that isolate JR02 (GenBank accession OP692731) was 100% identical to R. stolonifer (GenBank accession MT256940). In pathogenicity tests,10 healthy young fruit at a similar maturity stage as the ones found diseased were collected from a orchard where the disease was not observed. Fruit were surface sterilized with 70% ethylalcohol and washed with sterile distilled water. Wounded (using a sterilized needle) and non-wounded fruits were inoculated with 20µl of a spore suspension (1×106/ml). Sterile distilled water was used for the controls. Inoculated fruit were covered with sterile cloth, transferred to perforated plastic bags with moistened blotting paper, and incubated at 25°C in the dark. Symptoms were first observed after 2 days on wounded fruit, but no symptoms developed on controls and non-wounded fruit. Rhizopus stolonifer was re-isolated from infected fruit, thus fulfilling Koch's postulates. Rhizopus rot is a devastating disease causing premature fruit drop, reduced crop yield, and post-harvest rot of jackfruit and other fruits and vegetables (Sabtu et al. 2019). Three Rhizopus species namely R. stolonifer, R. artocarpi and R. oryzae have been reported causing fruit rot of jackfruit in the tropics including Mexico, India and Hawaii (García-Estrada et al., 2019; Babu et al., 2018; Nelson, 2005). Appropriate management strategies are needed to be developed to prevent premature rot of jackfruit. To our knowledge, this is the first report of R. stolonifer causing premature soft rot of jackfruit in Bangladesh.