The Use of the PCR-Based Dot-Blot Hybridization Assay to Detect Resistance Markers to Rifampicin and Streptomycin in Mycobacterium tuberculosis Isolates from the SW Region of Cameroon

Drug sensitivity testing to establish resistance to TB drugs takes many months to arrive at. Public health physicians have difficulties with such an approach due to long wait periods and cannot use it to establish community wide prevalence as a way to understand where resistance may be emerging faster and to limit its spread. The objective of this study was to use the dot-blot hybridization technique in the detection of resistance to rifamycin (RIF) and streptomycin (SM) in South- Western Cameroon and to compare the technique with the routine culture and drug susceptibility testing for detecting resistance in a resource poor country, Cameroon. A hospital-based study was conducted at the Regional hospitals of Buea and Limbe and Tiko Central Clinic. Tuberculosis (TB) patients aged 15 to 50 (mean age: 30.50 ± 8.33 standard deviation) were recruited for the study between December 2006 and April 2007. Cultures from 59 patients were tested for rifampicin and streptomycin sensitivity by the modified proportion method and mutational analysis for rpoB codon 516 and rrs codon 513 was performed by the dot-blot hybridization technique. Of the 59 sputum samples collected (36 were males and 23 were females) came from Buea 19 (32.2%), Limbe 20 (33.9%) and Tiko 20 (33.9%) towns respectively. Amplification for the gene showed that there was (59) 100% amplification with primers used for rpoB genes and 43 (72.9%) amplification with primers used for the rrs gene. Mutational analysis demonstrated that resistance to RIF was common in females (52.1%) than males (41.7%) while 6% of the samples were indeterminate. 12 (20.3%) samples showed phenotypic and genotypic resistance to RIF compared to 34 samples (58.1%) for SM. Phenotypic resistance and genotypic susceptibility were found in 5 (8.5%) RIF and 3 (4.7%) SM compared to phenotypic susceptibility and genotypic resistance that were found in 2 (3.5%) RIF and 3(4.7%) SM. Double mutation on rpoB and rrs genes occurred in 8 (13.6%) DNA samples. Resistance to RIF and SM due to mutations on the rpoB and rrs genes respectively in the SW region was found to be high and comparable to the drug susceptibility testing by 92%, (95% CI: 75.7 - 99.1). The Dot-blot technique will be useful in rapidly assessing the effectiveness of national TB control programs in limiting the spread of resistance strains in Cameroon.

An easy PCR-based genome-walking method for getting the unknown 5’flanking region of a Scenedesmus sp.

Objective:To develop the current single primer PCR-based genome-walking method with Scenedesmus sp.Methods:The unknown 5’and/or 3’flanking regions for a specific conserved sequence were optimized and the current single primer PCR-based genome-walking method were developed.Alignment was between the related species of microalga and Scenedesmus sp.For 18S rDNA,we selected the species Scenedesmus sp.,Chlorella sp.,and Chlamydomonas sp.For the rbcL gene from the chloroplast genome,alignment was done between Scenedesmus sp.,and Chlamydomonas sp.Results:Obtaining a small conserved sequence for any gene family is something that can be achieved quite easily.However,identifying the whole gene is often difficult.After investigating and testing,some of the current protocols using to get the unknown 5’and/or 3’flanking regions for a specific conserved sequence,we developed the current single primer PCR-based genome-walking method.We performed two consecutive PCR reactions;band extraction and the PCR product were sequenced.We got our results by testing the method on three genes from the total DNA of Scenedesmus sp.;two genes had a fully known sequence in gene bank(18S rDNA and rbcL),but the third one has not yet been identified(rbcS).We designed our primers based on the alignment between the related species and to each other.We also tested two different DNA polymerases Ex Taq and TLA polymerase.Conclusions:Results from our study suggest that Ex Taq is the most suitable polymerase for the current protocol.

Recent advances and perspectives of nucleic acid detection for coronavirus

The recent pneumonia outbreak caused by a novel coronavirus(SARS-CoV-2)is posing a great threat to global public health.Therefore,rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments,saving people's lives and preventing epidemics.It is important to establish a quick standard diagnostic test for the detection of the infectious disease(COVID-19)to prevent subsequent secondary spread.Polymerase chain reaction(PCR)is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity.Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler opera-tion.A variety of improved or new approaches also have been developed.This review summarizes the currently available detection methods for coronavirus nucleic acid.It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coro-navirus infection.

Construction of a high-density adzuki bean genetic map and evaluation of its utility based on a QTL analysis of seed size

Adzuki bean(Vigna angularis(Willd.)Ohwi&Ohashi)is an annual cultivated leguminous crop commonly grown in Asia and consumed worldwide.However,there has been limited research regarding adzuki bean genetics,which has prevented the efficient application of genes during breeding.In the present study,we constructed a high-density genetic map based on whole genome re-sequencing technology and validated its utility by mining QTLs related to seed size.Moreover,we analyzed the sequences flanking insertions/deletions(In Dels)to develop a set of PCR-based markers useful for characterizing adzuki bean genetics.A total of 2904 markers were mapped to 11 linkage groups(LGs).The total length of the map was 1365.0 cM,with an average distance between markers of 0.47 cM.Among the LGs,the number of markers ranged from 208(LG7)to 397(LG1)and the total distance ranged from 97.4 cM(LG9)to 155.6 cM(LG1).Twelve QTLs related to seed size were identified using the constructed map.The two major QTLs in LG2 and LG9 explained 22.1 and 18.8%of the total phenotypic variation,respectively.Ten minor QTLs in LG4,LG5 and LG6 explained 3.0–10.4%of the total phenotypic variation.A total of 9718 primer pairs were designed based on the sequences flanking In Dels.Among the 200 selected primer pairs,75 revealed polymorphisms in 24 adzuki bean germplasms.The genetic map constructed in this study will be useful for screening genes related to other traits.Furthermore,the QTL analysis of seed size and the novel markers described herein may be relevant for future molecular investigations of adzuki bean and will be useful for exploiting the mechanisms underlying legume seed development.

Cytogenetic identification and molecular marker development for the novel stripe rust-resistant wheat- Thinopyrum intermedium translocation line WTT11

Stripe rust,caused by Puccinia striformis f.sp.tritici(Pst),is one of the most destructive diseases of wheat(Triticum aestivum L)worldwide.Xiaoyan 78829,a partial amphidiploid developed by crossing common wheat with Thinopyrum intermedium,is immune to wheat stripe rust.To transfer the resis-tance gene of this excellent germplasm resource to wheat,the tr anslocation line WTT11 was produced by pollen irradiation and assessed for immunity to stripe rust races CYR32,CYR33 and CYR34.A novel stripe rust-resistance locus derived from Th.intermedium was confirmed by linkage and diagnostic marker analyses.Molecular cytogenetic analyses revealed that WTT11 carries a TTh 2DL translocation.The breakpoint of 1B was located at 95.5 MB,and the alien segments were found to be homoeologous to wheat-group chromosomes 6 and 7 according to a wheat660K single-nucleotide polymorphism(SNP)array analysis.Ten previously developed PCR-based markers were confirmed to rapidly trace the alien segments of WTT11,and 20 kompetitive allele-specific PCR(KASP)markers were developed to enable genotyping of Th.intermedium and common wheat.Evaluation of agronomic traits in two consecutive crop seasons uncovered some favorable agronomic traits in WTT11,such as lower plant height and longer main panicles,that may be applicable to wheat improvement.As a novel genetic resource,the new resistance locus may be useful for wheat disease-resistance breeding.