Specific Primer Sets Research Articles

First report of perilla mosaic emaravirus infecting Perilla frutescens in South Korea.

Perilla mosaic virus (PerMV; the genus Emaravirus in the family Fimoviridae) has a multiple, negative-sense, single-stranded RNA genome (ICTV, 2018). PerMV has been reported in Japan, where it was transmitted by an eriophyid mite species (Acari: Eriophyidae) to Perilla frutescens (L.) Britton var. crispa (Kubota et al., 2020). In September 2021, typical symptoms of the virus including yellow flecks, mosaic symptoms, and malformation were observed in leaves of P. frutescens in a cultivated field in Iseo-myeon, Wanju, South Korea (Suppl. Fig. 1). Visual estimates indicated that symptom incidence reached 70%, and the top leaves of perilla plants exhibited more severe symptoms and leaf distortion. To identify the virus species accurately, total RNA was extracted from five symptomatic perilla leaves collected using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) then cDNAs were amplified by reverse-transcription polymerase chain reaction (RT-PCR) using two pairs of primers to PerMV specific primer set designed to amplify 412- and 491-bp cDNAs of the nucleocapsid protein gene RNA 3 and movement protein gene RNA 4, respectively (Suppl. Table). Single-infection of PerMV in symptomatic Korean perilla plants was confirmed by high-throughput sequence (HTS) analysis and de novo transcriptome assembly using the Illumina HiSeq 4000 platform (Macrogen Inc., Seoul, Korea). The assembled sequences were aligned with viral reference genomes through searches performed using the BLASTn tool. Seven contigs (597-7,213 bp) revealed 92.09-97.37% nucleotide homology with RNAs of the isolate PerMV_Kochi_Nankoku_2011 (accession numbers LC496090 to LC496099) in the GenBank database. Other viruses including turnip mosaic virus and cucumber green mottle mosaic virus were not identified by HTS analysis (Cho et al., 2021; Park et al., 2020; Song et al., 2022). Seven RNA genomes of PerMV were confirmed by RT-PCR using specific primer sets designed to amplify part of each genome (Suppl. Table 1 and Fig. 2). The complete nucleotide sequences of PerMV (named IS isolate) RNA 1-7 were determined to be 7,177, 2,089, 1,094, 1,302, 1,079, 1,098, and 995 bp in length, respectively; these were deposited in GenBank (LC721296-LC721303). Sap from a symptomatic leaf sample confirmed for single infection was inoculated mechanically onto the leaves of 10 healthy P. frutescens seedlings, which developed the same PerMV symptoms within 3 weeks. These results indicate that PerMV is the causal agent of viral disease in Korean perilla plants cultivated in South Korea. To our knowledge, this is the first report of a perilla mosaic emaravirus infecting to Korean perilla, P. frutescens in South Korea.

Molecular Identification of Ribbon Fish (Eupleurogrammus Sp.) Using Partial Sequence of Mitochondrial COI Gene

Dry fish is considered as a delicacy in the menu of many people of Bangladesh. The most economically important marine and freshwater dried fishes are ribbon fish, bombay duck, olive barb, pomfret, shrimp, etc. In this study, fresh ribbon fish sample was collected from Cox's Bazar and morphologically identified as ribbon fish. The collected fish was confirmed through a molecular technique using mitochondrial COI gene (cytochrome c oxidase subunit I) to avoid morphological ambiguity, which is first reported data from Bangladesh. Partial sequence of this COI gene was amplified using a genus specific universal primer set. The target nucleotide sequence of COI gene was determined successfully with 616 bp length and the ribbon fish was identified as Eupleurogrammus sp. Within the amplified region GC and AT content were 45.5% and 54.5%, respectively. 16.88% interspecific polymorphism was observed when compared with other species under genus Eupleurogrammus collected from Gen Bank database. This sequence will be used as molecular bar code for authentic identification of Eupleurogrammus sp. at genus level. Bangladesh J. Zool. 50 (2): 231-238, 2022

Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2).

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

1-Kestose Supplementation Increases Levels of a 5α-Reductase Gene, a Key Isoallolithocholic Acid Biosynthetic Gene, in the Intestinal Microbiota.

1-Kestose (kestose) is the smallest fructooligosaccharide component and shows a particularly high prebiotic function. Both kestose and the bile acid metabolite isoallolithocholic acid (isoalloLCA) are known to be beneficial for human health, especially in terms of immune homeostasis in the gastrointestinal system; however, the effect of kestose on the levels of microbial isoalloLCA producers remains to be clarified. IsoalloLCA is known to be produced by several members of the phylum Bacteroidota that carry the 5α-reductase (5AR) gene, a key isoalloLCA biosynthetic gene. Thus, we designed a specific primer set to detect the 5AR gene based on the consensus sequences of the genes from several isoalloLCA producers. Using real-time quantitative PCR with this primer set and fecal DNA samples, we compared the 5AR gene level (5ar-level) in the intestinal microbiota of a kestose-supplemented group (n=20) and a placebo group (n=16) before and after intake for 12 wk. The 5ar-level was significantly increased in the kestose-supplemented group (p=0.015), but not in the placebo group (p=0.379), indicating that kestose supplementation increased the 5ar-level in human intestinal microbiota. Our findings suggest that targeting functional gene levels could potentially be used to predict and understand the beneficial prebiotic effects associated with changes in gut microbiota.

Mapping the Universe of Eph Receptor and Ephrin Ligand Transcripts in Epithelial and Fiber Cells of the Eye Lens.

The eye lens is a transparent, ellipsoid organ in the anterior chamber of the eye that is required for fine focusing of light onto the retina to transmit a clear image. Cataracts, defined as any opacity in the lens, remains the leading cause of blindness in the world. Recent studies in humans and mice indicate that Eph–ephrin bidirectional signaling is important for maintaining lens transparency. Specifically, mutations and polymorphisms in the EphA2 receptor and the ephrin-A5 ligand have been linked to congenital and age-related cataracts. It is unclear what other variants of Ephs and ephrins are expressed in the lens or whether there is preferential expression in epithelial vs. fiber cells. We performed a detailed analysis of Eph receptor and ephrin ligand mRNA transcripts in whole mouse lenses, epithelial cell fractions, and fiber cell fractions using a new RNA isolation method. We compared control samples with EphA2 knockout (KO) and ephrin-A5 KO samples. Our results revealed the presence of transcripts for 12 out of 14 Eph receptors and 8 out of 8 ephrin ligands in various fractions of lens cells. Using specific primer sets, RT-PCR, and sequencing, we verified the variant of each gene that is expressed, and we found two epithelial-cell-specific genes. Surprisingly, we also identified one Eph receptor variant that is expressed in KO lens fibers but is absent from control lens fibers. We also identified one low expression ephrin variant that is only expressed in ephrin-A5 control samples. These results indicate that the lens expresses almost all Ephs and ephrins, and there may be many receptor–ligand pairs that play a role in lens homeostasis.

Interleukin 10 Gene Promoter Polymorphisms in Patients with Chronic Periodontitis

Background: The anti-inflammatory cytokine, interleukin 10 (IL 10), has a crucial role in periodontal diseases. The gene coding for this cytokine is pleomorphic. In the promoter region of the IL 10 gene, two single nucleotide polymorphisms (SNPs), IL10 1082 and IL 819, have been discovered to affect IL 10 expressions. Objective: To investigate the association of two single nucleotide polymorphisms in the interleukin 10 gene (IL-10-1082 G/A and IL-10-819 C/T) with CPO. Patients and Methods: A total of 64 patients with chronic periodontitis (CPO) and another 40 apparently healthy individuals were enrolled in this case-control study. The IL-10 gene was amplified with two specific sets of primers. Genotyping was achieved by allele-specific polymerase chain reaction (AS-PCR). Results: The frequency of GG, GA and AA genotypes of the SNP IL-10-1082G/A among patients was 48.44%, 42.19% and 9.37% respectively, compared with 32.5%, 35% and 32.5% respectively, among controls. Logistic regression test revealed a significant difference in the frequency of AG (OR=0.19, 95%CI=0.06-0.62, P=0.006) and GG genotype (OR= 0.24, 95%CI=0.07-0.77, p=0.016) between patients and controls. Furthermore, the haplotype block GC (G allele of IL-10-1082G/A and C allele of IL-10-819 C/T) was more common in controls than patients (36.25% vs. 33.44%) with a significant difference (OR= 0.54, 95%CI= 0.29-0.99, p= 0.048). Conclusion: These data suggest that the IL-10-1082 G/A gene polymorphism could be a risk factor for CPO in Iraqi patients.

Interleukin 10 Gene Promoter Polymorphisms in Patients with Chronic Periodontitis

Background: The anti-inflammatory cytokine, interleukin 10 (IL 10), has a crucial role in periodontal diseases. The gene coding for this cytokine is pleomorphic. In the promoter region of the IL 10 gene, two single nucleotide polymorphisms (SNPs), IL10 1082 and IL 819, have been discovered to affect IL 10 expressions.
 Objective: To investigate the association of two single nucleotide polymorphisms in the interleukin 10 gene (IL-10-1082 G/A and IL-10-819 C/T) with CPO.
 Patients and Methods: A total of 64 patients with chronic periodontitis (CPO) and another 40 apparently healthy individuals were enrolled in this case-control study. The IL-10 gene was amplified with two specific sets of primers. Genotyping was achieved by allele-specific polymerase chain reaction (AS-PCR).
 Results: The frequency of GG, GA and AA genotypes of the SNP IL-10-1082G/A among patients was 48.44%, 42.19% and 9.37% respectively, compared with 32.5%, 35% and 32.5% respectively, among controls. Logistic regression test revealed a significant difference in the frequency of AG (OR=0.19, 95%CI=0.06-0.62, P=0.006) and GG genotype (OR= 0.24, 95%CI=0.07-0.77, p=0.016) between patients and controls. Furthermore, the haplotype block GC (G allele of IL-10-1082G/A and C allele of IL-10-819 C/T) was more common in controls than patients (36.25% vs. 33.44%) with a significant difference (OR= 0.54, 95%CI= 0.29-0.99, p= 0.048).
 Conclusion: These data suggest that the IL-10-1082 G/A gene polymorphism could be a risk factor for CPO in Iraqi patients.

Establishment of transgenic potato cultivar IPB CP1 plants containing gene encoding for superoxide dismutase to increase the abiotic stress tolerance

Potato ( Solanum tuberosum L.) cultivar IPB CP1 is suitable as a raw material for the potato chip industry. Potato plants are sensitive to various abiotic stresses such as drought, aluminium and salinity, which induce reactive oxygen species (ROS). ROS is very toxic to plant cells. Superoxide dismutase (SOD) is one of the enzymes that catalyse ROS to H2O2 and O2. This study aimed to establish transgenic potato cv. IPB CP1 plants containing the MmCuZn‐SOD gene that are tolerant to various abiotic stresses. Genetic transformation using internodes without buds as explants produced putative transgenic potato with a transformation efficiency of 51.25% and a regeneration efficiency of 38.87%. Integration analysis of the MmCuZn‐SOD transgene in putative transgenic plants by polymerase chain reaction (PCR) with a set of specific primers showed that eight plants contained the MmCuZn‐SOD gene under the control of the 35S CaMV promoter. In vitro salinity stress, aluminium stress, and drought stress assays showed that transgenic plants had a higher number of roots and total root length than non‐transgenic ones. These results indicate that transgenic potato cv. IPB CP1 plants are more tolerant to abiotic stresses than non‐transgenic ones.

S8.5c MLST genotyping and phylogenetics of AD-hybrids

S8.5 Genotyping of Cryptococcus neoformans and C. gattii, September 23, 2022, 3:00 PM - 4:30 PMObjectivesIn a previous study a set of new molecular-type specific primers were designed to apply the standard ISHAM consensus multi-locus sequence typing (MLST) scheme to Cryptococcus neoformans AD hybrids. In the present study, we report the preliminary results of the investigation by MLST of a large number of AD hybrids with the aim to identify the circulating genotypes, their phylogenesis, and population genetics.MethodsA total of 50 AD-hybrid isolates from different parts of the world and from different sources were genotyped by MLST. Minimum spanning trees using GoeBurst algorithm were generated by comparing hybrid genotypes and by comparing separately either allele-A and allele-D portions of the hybrid genotypes to the haplotypes recorded in the MLST global database.ResultsAnalysis identified 32 hybrid genotypes grouped in three distinct main clusters (CC12, CC21, and CC30) including 12 isolates each. Both CC12 and CC21 clusters included isolates from different countries and continents but the former grouped only isolates with mating type aADalpha whereas the latter those with mating type alphaADa. Cluster CC30 included only isolates from Ivory Coasts. Heterozygous allelic combinations in each of the seven MLST loci presented two or three combinations more frequent than the other ones. In some isolates, one or more alleles were not amplified after multiple attempts, and therefore, they were considered as lacking. A total of 22 MLST profiles were identified by analyzing separately the allele-A combinations of the hybrids. Comparison with all MLST profiles of VNI, VNII, and VNB included in the MLST global database showed that the allele-A portion of the hybrid genotypes was grouped in few VNI or VNB clusters. In none of the investigated hybrids, the allele-A portion originated from VNII genotypes. Similarly, when the MLST profile of allele-D portion of hybrids was compared to all VNIV genotypes present in the global MLST database, few clusters were identified but, in this case, mostly originated from genotypes not yet found among VNIV haplotypes.ConclusionsThese preliminary results suggest that the AD hybrids here investigated originated from the mating of A haploids very common in both clinical and environmental isolates and D haploids that are not circulating at present or are very rare. Therefore, it is likely that hybrids originated in the environment where VNIV genotypic diversity is higher and suitable AD combinations can occur. Sequencing of further AD hybrids is in progress to confirm these results.

A SYBR Green qPCR assay for specific detection of Colletotrichum ocimi, which causes black spot of basil

Colletotrichum ocimi causes black spot of basil (Ocimum basilicum) and is a serious threat to basil cultivation as it compromises leaf production. The pathogen also infects seeds, which could become primary sources of inoculum for spread of black spot. A SYBR Green real-time PCR assay was developed to detect Colletotrichum ocimi in basil leaves and seeds, based on the partial β-tubulin (tub2) gene sequence. Two primer sets were designed and tested. The selected primer pairs produced amplicons of 130 bp. The real-time PCR assay was validated for analytical specificity, sensitivity, selectivity, repeatability and reproducibility. The assay was specific for C. ocimi with respect to ten Colletotrichum spp. and to another 12 pathogens of basil plants. Sensitivity was 1 pg µL-1 of genomic fungal DNA and amplification analyses were not influenced by basil genomic DNA. The assay detected and quantified C. ocimi in artificially inoculated basil leaves. This is the first specific primer set for C. ocimi, which allows rapid detection and quantification of the pathogen is a useful tool for diagnostics in plants. Detection in seeds would also be possible, but will require an optimized extraction method. The qPCR detection of C. ocimi in planta can contribute to adoption of effective preventive disease management strategies.

Association Of Hepcidin (HAMP) Gene Polymorphism And Cytokines, Iron, And Hb Levels In Covid-19 Patients

Introduction: COVID-19 is a new type of coronavirus that infects humans and causes severe acute respiratory syndrome (SARS). Isolated in Wuhan City, China, in December 2019. Similar to discoveries relating to the coronaviruses that cause severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Levels of cytokines (Interleukin-6, Interleukin-1 β ), iron and hemoglobin changed during COVID-19 disease. Hepcidin is an iron regulatory protein that is encoded by the HAMP gene . 
 Material and Methods : Serum IL-6, IL-1 β , Hepcidin are measured by an ELISA kit and Iron ,UIBC , TIBC are measured by colorimetric methods. The genotyping was done by the ARM-PCR technique with a specific set of primers. 
 Results : The difference in Hb and serum iron levels, UIBC, TIBC, IL-1, IL-6, and Hepcidin conc. between the patient and recovery groups is statistically significant at P≤ 0.05 . No significant difference between the two groups in hepcidin gene polymorphism because of Polymorphisms of the hepcidin gene appeared in the two groups (in most samples). 
 Conclusions: The persons involved in this study are (most of them) heterozygous, and this indicates the presence of the mutation allele in the two groups , and it may be one of the reasons for the deficiency in hepcidin that appeared in this study.

From Clinical Specimen to Whole Genome Sequencing of A(H3N2) Influenza Viruses: A Fast and Reliable High-Throughput Protocol.

(1) Background: Over the last few years, there has been growing interest in the whole genome sequencing (WGS) of rapidly mutating pathogens, such as influenza viruses (IVs), which has led us to carry out in-depth studies on viral evolution in both research and diagnostic settings. We aimed at describing and determining the validity of a WGS protocol that can obtain the complete genome sequence of A(H3N2) IVs directly from clinical specimens. (2) Methods: RNA was extracted from 80 A(H3N2)-positive respiratory specimens. A one-step RT-PCR assay, based on the use of a single set of specific primers, was used to retro-transcribe and amplify the entire IV type A genome in a single reaction, thus avoiding additional enrichment approaches and host genome removal treatments. Purified DNA was quantified; genomic libraries were prepared and sequenced by using Illumina MiSeq platform. The obtained reads were evaluated for sequence quality and read-pair length. (3) Results: All of the study specimens were successfully amplified, and the purified DNA concentration proved to be suitable for NGS (at least 0.2 ng/µL). An acceptable coverage depth for all eight genes of influenza A(H3N2) virus was obtained for 90% (72/80) of the clinical samples with viral loads >105 genome copies/mL. The mean depth of sequencing ranged from 105 to 200 reads per position, with the majority of the mean depth values being above 103 reads per position. The total turnaround time per set of 20 samples was four working days, including sequence analysis. (4) Conclusions: This fast and reliable high-throughput sequencing protocol should be used for influenza surveillance and outbreak investigation.

Molecular detection and characterization of Potato spindle tuber viroid (PSTVd) infecting Tomato (Solanum lycopersicum L.) in Karnataka State of India.

Potato Spindle Tuber Viroid (PSTVd) is a non-coding, infectious, small, circular RNA known to cause disease in agricultural and horticultural plants. In the present work, an investigation was conducted in the southern districts of Karnataka state to assess the possible pospiviroid infections on tomato plants that are considered natural hosts for viroids. A total of 83 tomato samples showing disease symptoms (virus or viroid-like) along with healthy ones were collected and subjected to viroid detection by conventional Reverse Transcriptase Polymer Chain Reaction (RT-PCR) using universal (Pospi1-RE/Pospi1-FW) and a specific set of primers (3H1/2H1). The study confirmed the presence of PSTVd in one of the samples of tomato collected from Banghatta village of Mandy District, with an expected amplicon of ~ 361bp. The bioassay conducted on tomato plants (cv. Rutgers) proved the association of PSTVd, which was further confirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative sequences were deposited in the NCBI GenBank. The sequence alignment and secondary structure analysis of the isolated viroid with other reference sequences revealed the variations in the pathogenicity, central conserved region, and Terminal right domains. The variations observed between the isolated PSTVd with that of other Indian isolates support that viroid may have been transmitted among the crop plants, possibly through seed or mechanical means.

Bacterial diseases outbreaks in some freshwater fish farms in Kafr El-Sheikh, Egypt

ABSTRACT Bacterial fish diseases are one of the challenges facing the aquaculture industry and lead to tremendous economic losses. After surveying the infected farms understudy from April 2018 to November 2019, we collected 480 samples from 12 tilapia and carp farms experiencing mortalities in Kafr El-Sheikh region of Egypt with major aquaculture production. Clinically, hemorrhages, exophthalmia, and ulceration were the most common symptoms. We then determined the frequency, bacteriological and molecular identity, antibiotic sensitivity, pathogenicity, and histopathological characteristics of pathogens. For molecular identification was used a set of specific primers (16S rRNA, 16SPSEflu, and Sin-1b). Our results showed that Aeromonas hydrophila, Pseudomonas fluorescens, and Streptococcus iniae were the most important causes. Antibiotic sensitivity test revealed that all isolates were highly sensitive to ciprofloxacin. Additionally, naturally and experimentally infected fish are the same in pathognomonic signs. Overall, a valuable tool for diagnosis is the combination of both bacteriological and molecular examination. Besides, choosing the best antibiotics by sensitivity test helps avoid drug resistance phenomenon.

Design of Species-Specific Primers for Early Detection of Kretzschmaria zonata, the Causal Agent of Root and Neck Rot of Teak (Tectona grandis)

Kretzschmaria zonata (Lév.) P.M.D. Martin is a fungus that causes root and neck rot in teak (Tectona grandis L.) worldwide. The detection of this fungus in asymptomatic plants is essential for its control but, to date, the disease can only be detected morphologically or by sequencing this fungus’s isolates. The aim of this work was to design, at least, one set of specific PCR primers for a quickly, early and accurate diagnosis of this pathogen. Nineteen isolates of K. zonata from teak plants at different locations in Mexico were obtained. ITS region sequences and two software were used to design the PCR-based primers. All primers obtained were tested on DNA samples from infected teak tissue, K. zonata pure isolates, and other different fungi species (in vitro and in silico). The sensitivity of the primers was evaluated at five different concentrations of target DNA. The primer set KZ-AQ-3F/KZ-AQ-3R was selected for its specificity (amplifies only K. zonata) and sensitivity (1 × 10−5 ng/µL detection). This is the first report of a Kretzschmaria zonata-specific primer set. Tests carried out on asymptomatic teak and artificially inoculated plants with this fungus demonstrated the usefulness of these primers for the accurate detection of K. zonata, and taking early control measures.

Common polymorphisms of protein tyrosine phosphate non-receptor type 2 gene are not associated with risk of Crohn's disease in Indian.

BACKGROUNDMultiple genetic risk factors for Crohn’s disease (CD) have been identified. However, these observations are not consistent across different populations. The protein tyrosine phosphate non-receptor type 2 (PTPN2) gene plays a role in various aspects of host defense including epithelial barrier function, autophagy, and innate and adaptive immune response. Two common polymorphisms in the PTPN2 gene (rs2542151 and rs7234029) have been associated with risk of CD in Western countries. AIMTo evaluate the association of PTPN2 gene polymorphisms with risk of CD in Indian population.METHODSWe conducted a prospective case-control study. Patients with CD were recruited, and their clinical and investigation details were noted. Controls were patients without organic gastrointestinal disease or other comorbid illnesses. Two common polymorphisms in the PTPN2 gene (rs2542151 and rs7234029) were assessed. DNA was extracted from peripheral blood samples of cases and controls and target DNA was amplified using specific sets of primers. The amplified fragments were digested with restriction enzymes and the presence of polymorphism was detected by restriction fragment length polymorphism. The frequency of alleles was determined. The frequencies of genotypes and alleles were compared between cases and controls to look for significant differences.RESULTSA total of 108 patients with CD (mean age 37.5 ± 12.7 years, females 42.6%) and 100 controls (mean age 39.9 ± 13.5 years, females 37%) were recruited. For the single nucleotide polymorphism (SNP) rs7234029, the overall frequency of G variant genotype (AG or GG) was noted to be significantly lower in the cases compared to controls (35.2% vs 50%, P = 0.05). For the SNP rs2542151, the overall frequency of G variant genotype (GT or GG) was noted to be similar in cases compared to controls (43.6% vs 47%, P = 0.73). There were no significant differences in minor allele (G) frequency for both polymorphisms between the cases and controls. Both the SNPs had no significant association with age of onset of illness, gender, disease location, disease behaviour, perianal disease, or extraintestinal manifestations of CD.CONCLUSIONUnlike observation form the West, polymorphisms in the PTPN2 gene (rs7234029 and rs2542151) are not associated with an increased risk of developing CD in Indian patients.

Discrimination of Bacillus subtilis from Other Bacillus Species Using Specific Oligonucleotide Primers for the Pyruvate Carboxylase and Shikimate Dehydrogenase Genes.

Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

DESCRIPTION OF A NEW ISOLATE OF CUCUMBER MOSAIC VIRUS IN IRAQ

This study was initiated to isolate and characterize Cucumber mosaic virus based on serological, biological, and molecular approaches. Leaf samples were collected from symptomatic cucumber plants grown in protected fields at Plant protection Dept., College of Agricultural Engineering Sciences, the University of Baghdad at Al-Jhdryaa and used for biological assays. ELISA using CMV specific commercial kit was used to detect the virus in collected samples. RT-PCR using CMV specific primer set targeting CP gene was used to confirm CMV infection. DNA fragments amplified were sequenced and analyzed using computer software packages. Bioassays showed Vigna unguiculata exhibited necrotic local lesions when inoculated with cucumber samples. ELISA indicated that the cucumber sample was CMV infected scoring 2.907 highest absorbance at 405 nm. RT-PCR using CMV-F/ CMV-R primer set could amplify the targeted ~500 bp DNA fragments from cucumber samples only. Sequence analyses confirmed the detection when CMV isolated shared 95 and 91% maximum nucleotide (nt) and deduced amino acid (aa) sequence identities with the equivalent gene bank sequences of CMV CP from India, Japan, China, and USA. Neighbor-joining (NJ) phylogenetic tree, concentrated from partial CP gene nt and aa sequence, confirmed the relatedness when gouged CMV isolated to other CMV sequences suggesting a common origin. These findings confirmed the symptomatic cucumber sample collected from the protected culture was CMV infected.

Functional analysis of a matrix peptide involved in calcification of the exoskeleton of the kuruma prawn, Marsupenaeus japonicus

To clarify the calcification mechanism of the exoskeleton of marine crustaceans, kuruma prawns (Marsupenaeus japonicus), which are commercially important prawns in Japan, were used, and calcification-associated peptide (CAP), a matrix peptide that was previously reported in red swamp crayfish, was examined. Based on the RNA-sequencing data of kuruma prawns, a candidate Cap-1 cDNA sequence was found. Thereafter, the nucleotide sequence of kuruma prawn Cap-1 was determined by PCR methods using a specific primer set. Mature kuruma prawn CAP-1 showed a 66.7% amino acid identity with red swamp crayfish CAP-1. This sequence was presumed to be kuruma prawn CAP-1 because specific sequences, such as a chitin-binding consensus sequence, and many acidic amino acids in the C-terminal region were also observed in red swamp crayfish CAP-1. Then, a recombinant Cap-1 (rCAP-1) was expressed in Escherichia coli and purified by reverse-phase high-performance liquid chromatography. The purified rCAP-1 showed both chitin-binding and inhibitory activities of calcium carbonate precipitation. Based on Cap-1 mRNA expression analysis in the swimming legs and tail fans of juvenile prawns during molting, its mRNA expression level increased remarkably in the middle and late pre-molt stages. Cap-1-expressing cells were also identified by in situ hybridization using the swimming legs in the late pre-molt stage. It was found that kuruma prawn Cap-1 was expressed in epidermal cells under the cuticle layer. Due to knockdown using RNA interference (RNAi) methods, the Cap-1 mRNA expression in Cap-1-double-stranded RNA (dsRNA)-injected prawns decreased significantly in the middle and late pre-molt stages as compared with that in green fluorescent protein-dsRNA-injected control prawns. Furthermore, an irregularly shrunken and uneven structure appeared on the surface of the cuticle of dsRNA-Cap-1-treated prawns in the post-molt stage, and calcium deposition was inhibited by RNAi of Cap-1. Regarding marine crustaceans, to the best of our knowledge, this study is the first to determine that kuruma prawn CAP-1 has chitin-binding and anti-calcification activities and functionally changes its mRNA expression during molting. These results indicate that kuruma prawn CAP-1 plays important roles in exoskeleton calcification.

Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design.

BackgroundAs the rapidly evolving characteristic of SARS-CoV-2 could result in false negative diagnosis, the use of as much sequence data as possible is key to the identification of conserved viral sequences. However, multiple alignment of massive genome sequences is computationally intensive.ObjectiveTo extract conserved sequences from SARS-CoV-2 genomes for the design of diagnostic PCR primers using a bioinformatics approach that can handle massive genomic sequences efficiently.MethodsA total of 230,163 full-length viral genomes were retrieved from the NCBI SARS-CoV-2 Resources and GISAID EpiCoV database. This number was reduced to 14.11% following removal of 5′-/3′-untranslated regions and sequence dereplication. Fast, reference-based, multiple sequence alignments identified conserved sequences and specific primer sets were designed against these regions using a conventional tool. Primer sets chosen among the candidates were evaluated by in silico PCR and RT-qPCR.ResultsOut of 17 conserved sequences (totaling 4.3 kb), two primer sets targeting the nsp2 and ORF3a genes were picked that exhibited > 99.9% in silico amplification coverage against the original dataset (230,163 genomes) when a 5% mismatch between the primers and target was allowed. In addition, the primer sets successfully detected nine SARS-CoV-2 variant RNA samples (Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Iota, and Kappa) in experimental RT-qPCR validations.ConclusionIn addition to the RdRp, E, N, and S genes that are targeted commonly, our approach can be used to identify novel primer targets in SARS-CoV-2 and should be a priority strategy in the event of novel SARS-CoV-2 variants or other pandemic outbreaks.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13258-022-01264-7.