Dot Blot Hybridization Assay Research Articles

Thalassemia Genotypes and HbA2 levels of Children in Wuzhou, Guangxi

To analyze thalassemia genotypes and distribution of children in Wuzhou Guangxi, and evaluate the diagnostic value of HbA2 in children's thalassemia screening, so as to provide scientific evidence for the prevention and control strategies of thalassemia. Four hundred and fifty-eight children suspected with thalassemia in Wuzhou were enrolled from March 2017 to June 2022. The level of HbA2 was detected using Bio-Rad VARIANT II Hb analysis system. The deletion of α-thalassemia was measured with gap-PCR assay, and the point mutation of α- and β-thalassemia was tested with DNA reverse dot blot hybridization assay. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of HbA2 for children's thalassemia. A total of 304 thalassemia carriers were detected in 458 children, accounting for 66.38%. One hundred and seventy-five cases were defined to be α-thalassemia, with the main type of --SEA/αα (54.86%). Thirty-six cases were defined to be intermediate α-thalassemia, with the main type of -α3.7/--SEA (9.72%). In 108 cases with β-thalassemia, βCD41-42/βN was the main type, accounting for 49.07%, followed by βIVS-Ⅱ-654 /βN (14.81%). Seven cases were moderate/severe β-thalassemia (predominantly β-28/β-28 and βCD41-42/βCD17/). Twenty-one genotypes of α- and β-thalassemia were found in the children. There was significant difference of HbA2 level between the children with different types of thalassemia and healthy controls (all P < 0.001). ROC curve analysis showed that the sensitivities of HbA2 for α-thalassemia, β-thalassemia and αβ-thalassemia were 74.3%, 82.4% and 85.7%, with the optimal cut-off values of 2.60%, 3.60% and 3.70%, respectively, the specificities were 64.3%, 96.1% and 96.8%, and the area under the curve were 0.690, 0.887 and 0.916, respectively. The thalassemia genotypes of children in Wuzhou are diverse. It is necessary to further strengthen the prevention and control measure of thalassemia to reduce birth defects and improve birth quality.

Design of dot-blot hybridization assay for simultaneous detection of Campylobacter jejuni and Campylobacter coli: a preliminary study.

Campylobacters are a major cause of gastroenteritis worldwide. These are fastidious in culture and false negative results are seen in many clinical laboratories. Among molecular methods, the dot-blot technique is widely used for a variety of purposes, especially diagnostics. So, the authors aimed to detect C. jejuni and C. coli simultaneously using a dot-blot assay. After evaluating the bioinformatics studies, a cadF-conserved fragment was selected for the design of primers and probe. DNAs from standard strains and a recombinant plasmid, prepared in this study, were used to assess the technique. The specificity of the method was also surveyed using DNAs from other enteric bacteria. The limit of detection was evaluated by recombinant plasmid and different concentrations of the designed probe. A 95-bp fragment of cadF was selected, and in silico analysis studies showed that it is conserved between both species. Also, the non-specific annealing of the primers and probe with other bacteria was not seen theoretically. The technique with recombinant plasmid as well as DNAs of standard strains created black spots on the membrane, confirming that the probe was correctly synthesized. No non-specific reactions with other bacterial species were observed (specificity=100%). The limit of detection of the test was determined to be 50µg/ml. This is the first study to simultaneously detect two important pathogens in the Campylobacter genus and was able to detect C. jejuni and C. coli with acceptable sensitivity and specificity.

A universal probe for simultaneous detection of six pospiviroids and natural infection of potato spindle tuber viroid (PSTVd) in tomato in China

Several viroid species in the genus Pospiviroid can infect tomato (Solanum lycopersicum) and cause severe diseases, posing a serious threat to tomato production. For simultaneous detection of six tomato-infecting pospiviroids–columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd), and tomato planta macho viroid (TPMVd)–we developed a universal probe based on a highly conserved 61 nt long sequence shared among them. Compared with their specific probes, the universal probe had a similar, though slightly reduced, detection sensitivity and has the advantages of simple and cost-effective preparation and simultaneous detection of the six pospiviroids. In addition, the universal probe was used in dot-blot hybridization assays for a large-scale survey of viroid(s) in tomato plantings in China. Only PSTVd was detected in a few greenhouse-planted tomato plants. Sequence analysis revealed that these tomato PSTVd isolates may have been introduced from tomato seeds imported from abroad.

Head-to-head comparison of genotyping of human papillomavirus by GP5+/6+-PCR-based reverse dot blot hybridization assay and SPF10-PCR-based line probe assay.

The SPF10-PCR-based line probe assay (LiPA-25) for human papillomavirus (HPV) genotyping with high analytical sensitivity and specificity was widely used in HPV vaccine clinical trials and epidemiologic studies. In the study, we aimed to compare a novel GP5+/6+-PCR-based reverse dot blot hybridization assay (Yaneng-23) with LiPA-25. The performance of two assays was evaluated in 1735 cervical swab and 117 tissue samples, with a focus on 19 common HPV types (14 high-risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68/73; 5 low-risk: 6, 11, 42, 43, and 53). A total of 1197 (69.0%) swab samples were identified as HPV-positive by two assays. Of these, 878 (73.4%) samples displayed absolute agreement (concordant), 255 (21.3%) showed additional or fewer types (compatible), and the remaining 64 (5.3%) samples were discordant. Additionally, the two assays showed an excellent strength of agreement for 19 HPV-combined detection (κ = 0.886) and 17 individual HPV types (all κ > 0.800), and displayed a good agreement for HPV39 (κ = 0.780) and 42 (κ = 0.699). Yaneng-23 was more sensitive than LiPA-25 for HPV58, 59, 68/73, 42, 43 and 53 (McNemar's test: all p < 0.05), while LiPA-25 was more sensitive for HPV31, 39, 52, and 66 than Yaneng-23 (all p < 0.05). In 113 HPV-positive tissue specimens, the identification of genotypes was 82.3% identical and 17.7% compatible. The agreement between the tests for HPV45 (κ = 0.796) and 51 (κ = 0.742) was good, and for other types (all κ > 0.843) and 19 HPV-combined detection (κ = 0.929) was perfect (all p > 0.05). In conclusion, Yaneng-23 and LiPA-25 are comparable. Yaneng-23 could be used for the detection and genotyping of HPV in cervical samples for epidemiological and vaccine studies worldwide.

Reverse dot blot hybridization assay - An expeditious diagnostic tool for drug resistant TB.

Tuberculosis (TB) is one of the leading infectious diseases that is highly transmissible. It is reported to cause approximately 10 million infections and 1.4 million deaths globally in the year of 2019.¹ Prompt detection and treatment of TB are essential to intercept the proliferation of the disease. In the case of drug- resistant TB, failure to detect the resistance of anti TB drugs may give rise to extensively drug- resistant tuberculosis (XDR-TB). Thus, the rapid detection of drug resistance is vital;, however, the current standard drug susceptibility tests (DST) may take up to 12 weeks raising an alarming concern.² A study was done in China by Li Wan et al, on the accuracy of the reverse dot blot hybridization assay (RDBH) for rapidly detecting the resistance of four anti TB drugs (rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB)) in mycobacterium tuberculosis (MTB) isolates.³ Continuous...

CRISPR genotyping as complementary tool for epidemiological surveillance of Erwinia amylovora outbreaks.

Fire blight is a destructive plant disease caused by Erwinia amylovora affecting pome fruit trees, and responsible for large yield declines, long phytosanitary confinements, and high economic losses. In Portugal, the first major fire blight outbreaks occurred in 2010 and 2011, and although later considered eradicated, the emergence of other outbreaks in recent years stressed the need to characterize the E. amylovora populations associated with these outbreaks. In this regard, CRISPR genotyping, assessment of three virulence markers, and semi-quantitative virulence bioassays, were carried out to determine the genotype, and assess the virulence of thirty-six E. amylovora isolates associated with outbreaks occurring between 2010 and 2017 and affecting apple and pear orchards located in the country central-west, known as the main producing region of pome fruits in Portugal. The data gathered reveal that 35 E. amylovora isolates belong to one of the widely-distributed CRISPR genotypes (5-24-38 / D-a-α) regardless the host species, year and region. Ea 680 was the single isolate revealing a new CRISPR genotype due to a novel CR2 spacer located closer to the leader sequence and therefore thought to be recently acquired. Regarding pathogenicity, although dot-blot hybridization assays showed the presence of key virulence factors, namely hrpL (T3SS), hrpN (T3E) and amsG from the amylovoran biosynthesis operon in all E. amylovora isolates studied, pathogenicity bioassays on immature pear slices allowed to distinguish four virulence levels, with most of the isolates revealing an intermediate to severe virulence phenotype. Regardless the clonal population structure of the E. amylovora associated to the outbreaks occurring in Portugal between 2010 and 2017, the different virulence phenotypes, suggests that E. amylovora may have been introduced at different instances into the country. This is the first study regarding E. amylovora in Portugal, and it discloses a novel CRISPR genotype for this bacterium.

Application of a dot blot hybridization assay for genotyping Streptococcus uberis from Brazilian dairy herds

Streptococcus uberis is a major cause of environmental mastitis in many regions, and it is associated with clinical and subclinical infections. Although the main source of infection is the environment, reports of strains with a contagious profile have been described. Dot blot hybridization analysis allows the rapid identification of S. uberis population structures within and between herds, and it helps to identify strain diversity as well as possible clonal lineages that directly affect the control of bovine mastitis caused by this pathogen. The aim of this study was to evaluate the diversity of S. uberis isolates obtained from clinical (n = 22) and subclinical (n = 22) cases of mastitis in dairy herds (n = 13) in Brazil over a period of 12 mo. We submitted 44 S. uberis isolates to dot blot hybridization followed by automatic data analysis. We identified 8 different hybridization patterns using genetic markers associated with virulence factors and taxonomy, indicating diversity of S. uberis within the population and suggesting environmental transmission. However, the evidence of identical dot blot patterns in different mammary quarters from the same animal also suggested local contagious transmission. Of the virulence genes evaluated, we found a high prevalence of the genes sua, pauA, and gapC, highlighting the importance of these virulence factors for the adhesion, invasion, and multiplication of S. uberis in subclinical and clinical intramammary infections.

Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs Among Mycobacterium tuberculosis by a Reverse Dot Blot Hybridization Assay.

BackgroundReliable and timely determination of second-line drug resistance is essential for early initiation effective anti-tubercular treatment among multi-drug resistant (MDR) patients and blocking the spread of MDR and extensively drug-resistant tuberculosis. Molecular methods have the potency to provide accurate and rapid drug susceptibility results. We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization (RDBH) assay to simultaneously detect the resistance of fluoroquinolones (FQs), kanamycin (KN), amikacin (AMK), capreomycin (CPM) and second-line injectable drugs (SLIDs) in Mycobacterium tuberculosis.MethodsWe established and evaluated the accuracy of the RDBH assay by comparing to the phenotypic drug susceptibility testing (DST) and sequencing in 170 M. tuberculosis, of which 94 and 27 were respectively resistant to ofloxacin (OFX) and SLIDs.ResultsThe results show that, compared to phenotypic DST, the sensitivity and specificity of the RDBH assay for resistance detection were 63.8% and 100.0% for OFX, 60.0% and 100.0% for KN, 61.5% and 98.1% for AMK, 50.0% and 99.3% for CPM, and 55.6% and 100% for SLIDs, respectively; compared to sequencing, the sensitivity and specificity of the RDBH assay were 95.2% and 100.0% for OFX, 93.8% and 100.0% for SLIDs or KN (both based on mutations in rrs 1400 region and eis promoter), and 91.6% and 100.0% for AMK or CPM (both based on mutations in rrs 1400 region), respectively. The turnaround time of the RDBH assay was 7 h for testing 42 samples.ConclusionOur data suggested that compared to sequencing, the RDBH assay could serve as a rapid and reliable method for testing the resistance of M. tuberculosis against OFX and SLIDs, enabling early administration of appropriate treatment regimens among MDR tuberculosis patients.

Development of a novel polyprobe for simultaneous detection of six viruses infecting stone and pome fruits

A biotin-labeled, non-isotopic, novel polyprobe was developed for the simultaneous detection of six viruses viz. apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV), cherry virus A (CVA), prunus necrotic ringspot virus (PNRSV) and plum pox virus (PPV) infecting stone and pome fruit trees through dot-blot hybridization assay. The sensitivity of the polyprobe was checked by serial dilutions of total RNA extracted from the tissues of infected trees. ACLSV was detected up to a dilution of 5-5, whereas ApMV, ASGV, CVA, PPV and PNRSV up to 5-4. The developed assay was validated following testing a total of 45 symptomatic leaf samples collected from different geographical regions of Jammu and Kashmir (India), and the presence of the viruses was further confirmed by RT-PCR and sequencing. The polyprobe, designed for performing molecular hybridization assay can be developed quickly and avoid the tedious transformation and cloning procedures. Apart from simultaneously detecting viruses in stone and pome fruit trees, it holds great potential for virus indexing programmes to ascertain the supply of virus-free plant materials to the growers.

Cloning and expression of duck C4BPα and verification of its interaction with Riemerella anatipestifer

To study the interaction between C4b-binding protein (C4BP) and Riemerella anatipestifer (RA), we cloned duck C4BPα, conducted prokaryotic expression and prepared the polyclonal antibody by immunizing mice. Then indirect immunofluorescence assay and dot blotting hybridization assay were used to verify the interaction between C4BP and RA. The full length of duck C4BPα nucleotide sequence was 1 230 bp, with the highest similarity to chicken C4BPα (82.1%). Phylogenetic tree analysis showed that duck C4BPα and chicken C4BPα were on the same phylogenetic tree branch and the genetic evolution relationship between them was the closest. C4BPα was efficiently expressed in Escherichia coli BL21 (DE3). The recombinant proteins existed in intracellular soluble form. The titer of polyclonal antibody was more than 1:10 000 and polyclonal antibodies could specifically recognize the recombinant proteins. The results of indirect immunofluorescence assay and dot blot hybridization assay showed that RA could interact with duck C4BP. The results provide a basis to further reveal the pathogenesis of RA.

Accuracy of a reverse dot blot hybridization assay for simultaneous detection of the resistance of four anti-tuberculosis drugs in Mycobacterium tuberculosis isolated from China

BackgroundDrug resistant tuberculosis poses a great challenge for tuberculosis control worldwide. Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission of drug resistant Mycobacterium tuberculosis. We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization (RDBH) assay to simultaneously detect the resistance of four anti-tuberculosis drugs in M. tuberculosis isolated in China.MethodsIn this study, we applied a RDBH assay to simultaneously detect the resistance of rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) in 320 clinical M. tuberculosis isolates and compared the results to that from phenotypic drug susceptibility testing (DST) and sequencing. The RDBH assay was designed to test up to 42 samples at a time. Pearson’s chi-square test was used to compute the statistical measures of the RDBH assay using the phenotypic DST or sequencing as the gold standard method, and Kappa identity test was used to determine the consistency between the RDBH assay and the phenotypic DST or sequencing.ResultsThe results showed that the concordances between phenotypic DST and RDBH assay were 95% for RIF, 92.8% for INH, 84.7% for SM, 77.2% for EMB and the concordances between sequencing and RDBH assay were 97.8% for RIF, 98.8% for INH, 99.1% for SM, 93.4% for EMB. Compared to the phenotypic DST results, the sensitivity and specificity of the RDBH assay for resistance detection were 92.4 and 98.5% for RIF, 90.3 and 97.3% for INH, 77.4 and 91.5% for SM, 61.4 and 85.7% for EMB, respectively; compared to sequencing, the sensitivity and specificity of the RDBH assay were 97.7 and 97.9% for RIF, 97.9 and 100.0% for INH, 97.8 and 100.0% for SM, 82.6 and 99.1% for EMB, respectively. The turnaround time of the RDBH assay was 7 h for testing 42 samples.ConclusionsOur data suggested that the RDBH assay could serve as a rapid and efficient method for testing the resistance of M. tuberculosis against RIF, INH, SM and EMB, enabling early administration of appropriate treatment regimens to the affected drug resistant tuberculosis patients.

First Report of Tomato Chlorosis Virus Infecting Tectona grandis Associated With Infestation of Bemisia tabaci Mediterranean in Central Brazil

The commercial production of teak (Tectona grandis L. f., family Lamiaceae) has been increasing in Brazil due to the adaptation of this timber species to the predominant environmental conditions. Symptoms of interveinal yellowing in older leaves similar to that induced by species of the genus Crinivirus (family Closteroviridae) were observed (≈90% incidence) in 100-day-old teak seedlings in one orchard in Brasilia–DF in 2018. Moreover, a severe whitefly infestation was observed in association with these symptomatic plants. Fifteen leaf samples collected at random from distinct symptomatic plants were tested for the presence of tomato chlorosis virus (ToCV) and tomato infectious chlorosis virus (TICV), two criniviruses reported infecting a wide range of crops in Brazil (Fonseca et al. 2016). Leaf samples from asymptomatic teak plants (n = 10) were also collected and employed as negative controls. Total RNA extraction was performed using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) from all samples of symptomatic and asymptomatic plants. RT-PCR assays were conducted using the universal primer pair HS-11/HS-12 (which amplifies a 587-bp RNA-2 segment encompassing the HSP70h protein gene), followed by a nested-PCR performed with the primer pair ToCV- (ToC-5/ToC-6) and TICV-specific primers (TIC-3/TIC-4) (Dovas et al. 2002). All 15 symptomatic teak samples were positive only for ToCV (≈463-bp amplicons). PCR product from one randomly selected sample was directly sequenced. The obtained sequence (GenBank MH688047) displayed 99.7% identity with the equivalent sequence of a tomato-infecting ToCV isolate reported in Brazil (EU868927). Five symptomatic samples were also positive in dot-blot hybridization assays using a coat protein-derived RNA probe (436-nt fragment of RNA-2; primers MA-380/MA-381) (Fortes et al. 2012) labeled with digoxigenin (DIG)-11-UTP and with the chemiluminescent substrate CDP Star kit (Roche Diagnostics). Total DNA extraction and PCR assays were also conducted using pooled (three pools of n = 10 each) whitefly samples collected on symptomatic teak plants. A segment of the mitochondrial cytochrome oxidase I gene (mtCOI) was amplified using the specific primers C1-J-2195-FW and L2-N-3014- RV (Simon et al. 1994), yielding ≈860 bp amplicons. Direct sequencing of three amplicons obtained from the whitefly samples (MH688048) indicated high identity (99%) to B. tabaci Mediterranean (MED) species (= Q biotype). ToCV-transmission assays were performed using B. tabaci MED adults. Thirty viruliferous whiteflies derived from healthy whiteflies fed on ToCV-infected teak plants were confined in separated cages with healthy teak (n = 6) and tomato cv. Santa Clara (n = 6) seedlings. Aviruliferous whiteflies were also confined to healthy teak (n = 6) and tomato (n = 6) plants (negative controls). After 72 h, the whiteflies were eliminated by insecticide spraying. Thirty days after the feeding access period, only teak and tomato plants exposed to viruliferous B. tabaci MED exhibited crinivirus-like symptoms and were positive to ToCV in RT-PCR assays. To our knowledge, this is the first report of T. grandis as a host of ToCV as well as the first report of virus infection in this woody plant in Brazil. The introduction of B. tabaci MED into southern Brazil was recently reported (de Moraes et al. 2017). Our results also confirm the presence of B. tabaci MED in central Brazil and its association with ToCV infection of T. grandis seedlings. Although expressing overall mild symptoms, the potential negative effects of ToCV in teak plants need to be investigated in more detail.

Changes in methylation status of the whole genome and GCH1 gene in patients with herpes zoster neuralgia

Objective To analyze the correlation between herpes zoster neuralgia and the methylation status of the whole genome and GCH1 gene. Methods From June to October in 2017, patients with confirmed herpes zoster and obvious neuralgia were selected in Department of Dermatology, The Affiliated Hospital of Xuzhou Medical University, who achieved complete remission (no effect was observed on normal sleep) of neuralgia after antiviral and neurotrophic treatment. Finally, 36 patients and 36 healthy controls were enrolled into this study. Peripheral blood samples were obtained from the healthy controls and patients before and after the treatment. Dot-blot hybridization assay was performed to determine the methylation status of the whole genome, methylated-DNA IP kit was used to enrich the methylation sites of the GCH1 gene, and real-time quantitative PCR was conducted to detect changes in methylation status of the GCH1 gene. Statistical analysis was carried out with GraphPad Prism v7.00 software by using paired t test for the comparison of methylation status before and after the treatment, and two-sample t test for the comparison between the patient group and control group. Results The relative methylation level of the whole genome was 135.94 ± 2.52 in the patients before treatment, significantly lower than that in the patients after treatment (144.76 ± 3.48, t=2.056, P 0.05) . Compared with the patients after treatment (0.89 ± 0.13) and healthy control group (0.97 ± 0.07) , the methylation status of the GCH1 gene significantly decreased in the patients before treatment (0.65 ± 0.17; t=3.977, 4.648 respectively, P 0.05) . Conclusion The methylation status of the whole genome and GCH1 gene markedly decreased in the patients with herpes zoster neuralgia. Key words: Neuralgia, postherpetic; Methylation; Epigenomics; GCH1 gene

Identification of a novel linear B-cell epitope within the collagenase equivalent domain of porcine epidemic diarrhea virus spike glycoprotein

The porcine epidemic diarrhea virus (PEDV) collagenase equivalent domain (COE, residues 499–638), a crucial antigenic region within the viral spike (S) glycoprotein, has been widely utilized for the development of subunit vaccines to prevent viral infection. In the current study, we immunized BALB/c mice with recombinant truncated PEDV COE protein and obtained 14 COE-specific monoclonal antibodies (mAbs). Based on the reactivity analysis of the mAbs with two prevalent PEDV strains in G2 type and the attenuated CV777 strain in G1 type, 6 mAbs were selected for subsequent identification of COE mAb-binding epitopes. Dot-blot hybridization and enzyme-linked immunosorbent assays (ELISAs) identified the peptide 592TSLLASACTIDLFGYP607 as a novel linear B-cell epitope involved in binding of mAbs 4D8F10 and 6F3E3. Subsequently, alanine (A)-scanning mutagenesis demonstrated that residues 606Y, 605G and 604F were core residues involved in recognition. Importantly, this novel COE epitope, including core residues, is conserved among G1 and G2 type PEDV strains. Further experiment indicates that the mAbs 4D8F10 and 6F3E3 were suitable for PEDV detection via mAb binding to the conserved epitope. The current work actually provides potential uses for the development of diagnostic methods to detect PEDV.

One-step multiplex RT-PCR for simultaneous detection of four viroids from hop (Humulus lupulus L.)

Hop (Humulus lupulus L.) plants are hosts to several viroids; some of them can be highly aggressive, and their infection can manifest as complete plant dieback. Since molecular detection of multiple viroids can be time consuming and cost inefficient, a reliable one-step multiplex RT-PCR (mRT-PCR) was developed to detect simultaneously all four viroids infecting hops: Hop latent viroid (HLVd), Hop stunt viroid (HSVd), Apple fruit crinkle viroid (AFCVd) and Citrus bark cracking viroid (CBCVd). Several primer pairs were tested on different viroid variants from hops, citruses and grapevines, and from among them, specific primer pairs for detection of hop viroids were selected and confirmed in a single-tube assay. To improve mRT-PCR reliability and validate its effectiveness, nad5 and DRH1 genes were included as an internal control. The specificities of single and mRT-PCR assays for all four viroids were comparable. The sensitivity of mRT-PCR was compared with that of dot-blot hybridization and single RT-PCR assay on biolistically infected hop plants. The results show mRT-PCR to be more sensitive than the dot-blot and slightly less sensitive than the single RT-PCR assay. Furthermore, mRT-PCR was validated using field samples and a group of 135 hop plants, which are used in the certification scheme for planting material propagation, and the method proved to be robust, rapid and simple. Additionally, this approach can be applicable to similar methods of systematic surveys of emerging diseases and epidemiological studies.

Porcine Deltacoronavirus Engages the Transmissible Gastroenteritis Virus Functional Receptor Porcine Aminopeptidase N for Infectious Cellular Entry.

Identification of cellular receptors used by coronavirus (CoV) entry into the host cells is critical to an understanding of pathogenesis and to development of intervention strategies. The fourth CoV genus, Deltacoronavirus, evolutionarily related to the Gammacoronavirus, has just been defined recently. In the current study, we demonstrate that porcine aminopeptidase N (pAPN) acts as a cross-genus CoV functional receptor for both enteropathogenic porcine deltacoronovirus (PDCoV) and alphacoronovirus (AlphaCoV) (transmissible gastroenteritis virus [TGEV]) based upon three lines of evidence. First, the soluble S1 protein of PDCoV bound to the surface of target porcine cell lines known to express pAPN as efficiently as TGEV-S1, which could be blocked by soluble pAPN pretreatment. Second, both PDCoV-S1 and TGEV-S1 physically recognized and interacted with pAPN by coimmunoprecipitation in pAPN cDNA-transfected cells and by dot blot hybridization assay. Finally, exogenous expression of pAPN in refractory cells conferred susceptibility to PDCoV-S1 binding and to PDCoV entry and productive infection. PDCoV-S1 appeared to have a lower pAPN-binding affinity and likely consequent lower infection efficiency in pAPN-expressing refractory cells than TGEV-S1, suggesting that there may be differences between these two viruses in the virus-binding regions in pAPN. This study paves the way for dissecting the molecular mechanisms of PDCoV-host interactions and pathogenesis as well as facilitates future vaccine development and intervention strategies against PDCoV infection.IMPORTANCE The emergence of new human and animal coronaviruses is believed to have occurred through interspecies transmission that is mainly mediated by a species-specific receptor of the host. Among the four genera of the Coronavirinae, a couple of functional receptors for the representative members in the genera Alphacoronavirus and Betacoronavirus have been identified, whereas receptors for Gammacoronavirus and Deltacoronavirus, which are believed to originate from birds, are still unknown. Porcine coronaviruses, including the newly discovered porcine deltacoronavirus (PDCoV) associated with diarrhea in newborn piglets, have posed a serious threat to the pork industry in Asia and North America. Here, we report that PDCoV employs the alphacoronavirus TGEV functional receptor porcine aminopeptidase N (pAPN) for cellular entry, demonstrating the usage of pAPN as a cross-genus CoV functional receptor. The identification of the PDCoV receptor provides another example of the expanded host range of CoV and paves the way for further investigation of PDCoV-host interaction and pathogenesis.

PCR-reverse dot blot of the nucleotide signature sequences of matK for the identification of Mitragyna speciosa, a narcotic species

Four species of Mitragyna plants exist in Thailand, including Mitragyna speciosa, M. rotundifolia, M. hirsuta, and M. diversifolia. M. speciosa, or “Kratom” in Thai, is the only narcotic species that has been prohibited by law in Thailand. It causes addiction and sometimes fatalities when overdosed. The accurate detection of this species is needed in forensic justice for legal proceedings. In this study, we aim to develop an assay based on DNA barcoding for the detection of M. speciosa in forensic specimens. DNA barcodes of the matK, rbcL, psbA-trnH and ITS2 regions of the four Mitragyna species were established. The matK nucleotide signature site was selected as a target for the discrimination of M. speciosa, a narcotic species, from non-narcotics by using PCR-reverse dot blot (RDB) hybridization assay. This RDB assay is highly specific and selective for M. speciosa, costs little and is easily performed without advanced analytical equipment. This method is an alternative to DNA sequencing for the detection of M. speciosa in suspected samples. Using PCR-RDB to detect the polymorphisms of matK holds promise as a routine method to test for the presence of M. speciosa in forensic specimens.

Analysis of Common β-Thalassemia Mutations in North Vietnam

Available and flexible choice of methods for screening and detecting β-thalassemia (β-thal) can promote control of thalassemia in developing countries. In this study, two methods, the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and reverse dot-blot hybridization assays were developed to detect common β-thal mutations in 244 thalassemia patients and 152 healthy people in North Vietnam. The most common mutation was codon 26 (G>A), also known as Hb E (HBB: c.79G>A), accounting for 26.4% of the total studied chromosomes, followed by codons 41/42 (–TCTT) (HBB: c.126_129delCTTT) and codon 17 (A>T) (HBB: c.c.52A>T), accounting for 19.4 and 16.4%, respectively. In addition, codon 95 (+A) (HBB: c.c.287_288insA) that is known as the Vietnamese mutation, accounted for 0.6%. Moreover, the heterozygous state of the four mutations was also found in healthy people, of which Hb E was again the most common mutation with a frequency 3.0%. The results of this study provide available methods and indicative data for preventive and control strategies concerning the genetic diagnosis of thalassemia.

Molecular probes for identification of UDP-glucuronosyltransferase 1 gene polymorphisms for Gilbert's syndrome diagnosis

The T-3279G mutation in Phenobarbital Responsive Enhancer Modulein (PBREM), TA-insertion in the TATA box, creating the A(TA)7TAA motif instead of the A(TA)6TAA, and G211A mutation in coding exon 1, particularly in Asians, of human UGT1A1 (UDP-glucoronosyltransferase) gene are the three common polymorphisms of Gilbert’s syndrome. This article employed dot blot hybridization to detect the T-3279G, A(TA)6/7TAA and G211A mutation of UGT1A1 gene in 15 patients. In the dot blot hybridization assay, PCR products were denatured and spotted on positively charged nylon membranes instead of acrylamide-modified slides. Then three pairs of probes were labeled by digoxigenin and hybridized with the spotted array. After hybridization, the signal was visualized using TMB color development solution and all possible polymorphisms of the T-3279G, A(TA)6/7TAA and G211A in 15 Chinese patients with hyperbilirubinemia were successfully identified. The results of 15 samples by dotblot hybridization were consistent with the sequencing results. The data showed that dot blot hybridization assay is a quick and low cost method to detect UGT1A1 gene polymorphisms and helpful to diagnosis of Gilbert’s syndrome.

Detection of viruses in the exotic shrimp Penaeus vannamei Boone, 1931 cultured in India

Pacific white shrimp Penaeus vannamei Boone, 1931, is a recently introduced species in India. P. vannamei samples, collected from various shrimp farms of Karnataka, India were subjected to polymerase chain reaction (PCR) based detection of whitespot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), taura syndrome virus (TSV) and infectious myonecrosis virus (IMNV). Out of the 81 shrimp samples analysed, 41 samples (50.6%) were found positive for WSSV and four (4.9%) were positive for IHHNV. Among 41 WSSV positive samples, 20 (48.7%) samples were found positive for WSSV by 1st step PCR, while the remaining 21 (51.2%) samples were positive by nested PCR. WSSV positive samples were further confirmed by dot blot hybridisation assay. However, clinical signs/disease symptoms were not observed in any of the shrimp samples tested positive for the viruses.