Hybridization Polymerase Chain Reaction Research Articles

Analytical evaluation of the automated genotyping system (GenPlex) compared to a traditional real-time PCR assay for the detection of high-risk human papillomaviruses.

The detection of high-risk human papillomaviruses (HPVs) is crucial for early screening and preventing cervical cancer. However, the substantial workload in high-level hospitals or the limited resources in primary-level hospitals hinder widespread testing. To address this issue, we explored a sample-to-answer genotyping system and assessed its performance by comparing it with the traditional real-time polymerase chain reaction(PCR) method conducted manually. Samples randomly selected from those undergoing routine real-time PCR detection were re-analyzed using the fully automatic GenPlex® system. This system identifies 24 types of HPV through a combination of ordinary PCR and microarray-based reverse hybridization. Inconsistent results were confirmed by repeated testing with both methods, and the κ concordance test was employed to evaluate differences between the two methods. A total of 365 samples were randomly selected from 7259 women. According to real-time PCR results, 76 were high-risk HPV negative, and 289 were positive. The GenPlex® system achieved a κ value greater than 0.9 (ranging from 0.920 to 1.000, p < 0.0001) for 14 types of high-risk HPV, except HPV 51 (κ = 0.697, p < 0.0001). However, the inconsistent results in high-risk HPV 51 were revealed to be false positive in real-time PCR by other method. When counting by samples without discriminating the high-risk HPV type, the results of both methods were entirely consistent (κ = 1.000, p < 0.0001). Notably, the GenPlex® system identified more positive cases, with 73 having anHPV type not covered by real-time PCR, and 20 potentially due to low DNA concentration undetectable by the latter. Compared with the routinely used real-time PCR assay, the GenPlex® system demonstrated high consistency. Importantly, the system's advantages in automatic operation and a sealed lab-on-chip format respectively reduce manual work and prevent aerosol pollution. For widespread use of GenPlex® system, formal clinical validation following international criteria should be warranted.

Assessment of the effects of CNR1, FAAH and MGLL gene variations on the synthetic cannabinoid use disorder

Abstract Objectives Given that drug addiction occurs as a result of complex gene-environment interaction, a number of studies claimed that cannabinoid receptor 1 (CNR1), fatty acid amide hydrolase (FAAH), and monoacylglycerol lipase (MGLL) single nucleotide polymorphisms (SNPs) are associated with the risk of substance use disorders such as cannabis, opioids, and, methamphetamine. However, scientific research on genetic susceptibility to synthetic cannabinoid addiction is limited. In this population-based case-control study, we aimed to evaluate the genetic susceptibility to synthetic cannabinoid use disorder in terms of these three endocannabinoid system genes in the Turkish population. Methods 100 individuals diagnosed with synthetic cannabinoid use disorder according to Diagnostics and Statistical Manual of Mental Disorders-5 criteria and 100 healthy volunteers have recruited for the study. Genotyping of the CNR1 rs1049353, FAAH rs324420, and MGLL rs604300 SNPs was performed using Real-Time Polymerase Chain Reaction hybridization probes. Results The patient and control groups consist of 98 % male, 2 % female, 80 % male, and 20 % female individuals, respectively. The genotype distributions were consistent with Hardy–Weinberg equilibrium for all SNPs (p&gt;0.05). FAAH rs324420 and MGLL 604300 SNPs were genotyped for the first time in the Turkish population, and the variant allele frequencies were found as 0.205 and 0.085, respectively. Allele frequencies and genotype distributions CNR1 rs1049353, FAAH rs324420, and MGLL rs604300 SNPs were similar between the patient and control group (p&gt;0.05). Conclusions These results indicate that CNR1, FAAH, and MGLL gene polymorphisms do not influence the risk of synthetic cannabinoid use disorder in the Turkish population.

Production of Bovine Rotavirus VP6 Subunit Vaccine in a Transgenic Fodder Crop, Egyptian Clover (Berseem, Trifolium alexandrinum) that Elicits Immune Responses in Rabbit.

Group A rotavirus causes acute gastroenteritis in young ones of animals worldwide and is responsible for a high rate of their morbidity and mortality leading to huge economic losses. Developing affordable and safer vaccine on large scale is imperative to reach cattle population worldwide for the long-term control of diarrhea. Rotavirus middle capsid protein layer, VP6, is the most immunogenic and highly conserved protein that induces immune responses against rotavirus. In the present study, bovine group A rotavirus VP6 protein has been expressed for the first time in a highly nutritious and palatable forage crop, Trifolium alexandrinum, using Agrobacterium tumefaciens-mediated stable nuclear transformation. Transgenic nature of the shoots regenerated from cotyledon explants and rooted on hygromycin-containing medium was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qPCR), and protein expression and quantification by Western blot and enzyme-linked immune-sorbent assay (ELISA), respectively. The transformation efficiency of 2.10% was obtained. The highest amount of VP6 protein produced in a transgenic line was 402ng/g fresh weights (0.03% of total soluble protein). Oral feeding of transgenic leafy shoots expressing VP6 protein stimulated systemic immunity by inducing significantly higher titers of anti-VP6 serum IgG antibodies in rabbit to reduce rotavirus infection. These transgenic fodder plants offer safer vaccine produced on large scale at low cost with reduced regulatory issues to improve livestock's health and wealth. These plants would be used as alternative to the current live attenuated vaccines to protect young calves against rotavirus infection.

Using Proline Synthetase and BT gene to improve soybean (Glycine max) drought and insect resistance

AbstractIn agricultural production, developing a high yield with good quality and a stable yield are significant goals. To meet the aim of agriculture and produce high‐yielding, drought‐resistant and insect‐resistant varieties are preferred in the breeding of soybean. The pCAMBIA3301 plasmid was used as the base vector, and an overexpression vector containing the proline synthase gene Fu‐1 was recombined. The Fu‐1 gene was genetically transformed into the high‐generation (T7 generation) containing CryAb13‐1 transgene via the pollen tube channel method. Soybean was used to create a novel material that is insect resistant as well as drought‐resistant. Traditional polymerase chain reaction (PCR) identification was used to identify the transformed T2 generation plants, and Southern blot hybridization (SBH) and fluorescence quantitative PCR were used for detection. The test results showed that there were 10 positive plants in the T2 generation plants that were detected by common PCR. SBH results showed that Fu‐1 and CryAb13‐1 genes were integrated into soybean in the type of a single copy. The expression levels of Fu‐1 and CryAb13‐1 genes were variable in different tissues, according to the results of fluorescence quantitative PCR. In soybean roots, stems and leaves, the average expression levels of the Fu‐1 gene were 4.43, 1.86 and 2.51, respectively. CryAb13‐1 gene was expressed in roots. The average expression levels in roots, stem and leaf were 1.80, 1.38 and 5.28, respectively. The highest expression level was found in leaves in different soybean tissues. The indoor drought resistance identification showed that the water content, relative conductivity, POD activity, SOD activity, MDA and Pro content of the transformed plants after seven days of stress were significantly changed, and the drought resistance ability was significantly improved. Hence, we obtained a new soybean that can resist both drought and insect.

Carriage and distribution of serotypes Streptococcus pneumoniae in healthy vaccinated children for the year 2021 in Bulgaria

Streptococcus pneumoniae is one of the common causes of disease in children like pneumonia, otitis and meningitis. Pneumococci colonize asymptomatically the nasopharynx and could cause severe life-threatening illnesses. Since 2010, the 10-valent pneumococcal conjugate vaccine (PCV-10) has been included in Bulgaria for routine immunization of children from 6 weeks to 2 years. For the period 2011-2019 the coverage with it is considered to be 90%. The aim of the present study is to determine the frequency of carriers and common serotypes of S. pneumoniae from nasopharyngeal isolates of children in 2021, more than ten years after the introduction of PCV10 in Bulgaria. Nasopharyngeal swabs were taken from 220 vaccinated children, aged 5 months to 5 years old, collected from January 2021 to December 2021. Molecular detection methods were used – polymerase chain reaction (PCR) for detection and typing. By amplification of the lytA gene S. pneumoniae was detected in the taken nasopharyngeal isolates, and by amplification of the cpsA gene capsule abundance was determined. Typing was performed by PCR and allelic hybridization. The results show a total of 73 positive for Streptococcus pneumoniae samples (33.2%). The most common serogroup/serotypes determined in carriers was 19B/19C (15%) followed by serotypes/serogroups – 15B/15C (8%), 23B (8%) and 10F/10C (7, 1%). In conclusion from the presented data, we estimate a high percentage of carriers in Bulgarian children, characterized mainly by non-vaccine serotypes in the pneumococcal populations.

Tumor profiling of KRAS, BRAF, and NRAS gene mutations in patients with colorectal cancer: A Lebanese major center cohort study

BackgroundIn the era of precision medicine, treatment schemes for advanced Colorectal (CRC) disease include monoclonal antibodies which block the epidermal growth factor receptor (EGFR) implicated in tumor proliferation, invasion, migration and neovascularization. Resistance to these agents has been correlated with activating downstream mutations in KRAS, BRAF and NRAS genes, among others, leading to constitutive activation of the EGFR axis bypassing EGFR blockade. The assessment of tumor RASandBRAFmutational status has thus become standard clinical practice. While multiple investigations reported roughly mutations rates of 40% in KRAS, 7% in NRAS and 5–15 % in BRAF, numbers vary across different populations with limited data specifically from the Middle East. MethodsThis is a retrospective observational Laboratory information system (LIS) chart review of all the patients with pathologically confirmed colorectal carcinoma (CRC) or metastatic CRC who underwent KRAS, NRAS and/or BRAF mutational analysis testing at the Molecular Diagnostics Laboratory of the American University of Beirut Medical Center (AUBMC) from January 2012 to December 2018, inclusive. Data retrieved included the results of mutation testing performed for KRAS, NRAS and BRAF genes, the age, gender, and tumor location for each patient. Analysis of the mutations was performed using polymerase chain reaction (PCR) hybridization StripAssay® (ViennaLab, Vienna, Austria). Results130 (47.6%) out of 273 histologically confirmed CRC cases, had positive KRAS mutations, namely in codons 12 (82%), 13 (17%), 146 (1.5%), 117 (0.75%), or 61 (0.75%). Two patients had two concomitant mutations: 12 + 12 (different mutations) and 12 + 146. Of 203 CRC cases tested for NRAS mutations, 16 (7.8%) were found to be positive for a mutation in codon 12 (37.5%), 61 (37.5%), or 13 (12.5%). Two patients had two concomitant mutations: 12 + 13 and 59 + 61. Of 172 CRC cases tested for BRAF mutations, 2 (1.2 %) were positive for the V600E -. ConclusionThis retrospective study is the first to report the frequencies of KRAS, NRAS and BRAF gene mutations in a Lebanese CRC cohort diagnosed and managed at a tertiary care center. The frequencies of the studied somatic gene mutations were similar to previously reported cohorts in other populations however the rate of BRAF mutation was lower in this cohort than expected.

Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease Using Ac/Ds Transposon-Mediated Transgene Reintegration System.

Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.

Improved in planta genetic transformation efficiency in bitter gourd (Momordica charantia L.)

Production of transformed bitter gourd plants through in vitro regeneration is a laborious practice, which may also result in somaclonal variations. Thereby, in the current study, we have established a less tedious in planta protocol for more efficient genetic transformation of bitter gourd var. NBGH-470 (Apurva) using the seed as a target material. Bitter gourd seeds were transformed by Agrobacterium tumefaciens strain LBA4404 bearing the pCAMBIA1301 binary vector, and the transformed plants were selected against hygromycin B. The putatively transformed bitter gourd (To) plants were examined by GUS assay. Two parameters, including vacuum infiltration and sonication improving the in planta transformation, have been investigated in the present work. Amongst several time durations examined, the highest transformation rate (37%) was achieved upon subjecting the pre-cultured bitter gourd seeds to sonication for 15 min, followed by vacuum infiltration for 6 min in LBA4404 culture suspension. The transformed bitter gourd (T1) plants were selected against hygromycin B, and the transgene integration was evaluated by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase PCR (RT-PCR). The developed protocol in the current study is suitable to genetically engineer the bitter gourd plants with disease and pest-resistant traits.

Recent Advances in Molecular Diagnostics of Fungal Plant Pathogens: A Mini Review.

Phytopathogenic fungal species can cause enormous losses in quantity and quality of crop yields and this is a major economic issue in the global agricultural sector. Precise and rapid detection and identification of plant infecting fungi are essential to facilitate effective management of disease. DNA-based methods have become popular methods for accurate plant disease diagnostics. Recent developments in standard and variant polymerase chain reaction (PCR) assays including nested, multiplex, quantitative, bio and magnetic-capture hybridization PCR techniques, post and isothermal amplification methods, DNA and RNA based probe development, and next-generation sequencing provide novel tools in molecular diagnostics in fungal detection and differentiation fields. These molecular based detection techniques are effective in detecting symptomatic and asymptomatic diseases of both culturable and unculturable fungal pathogens in sole and co-infections. Even though the molecular diagnostic approaches have expanded substantially in the recent past, there is a long way to go in the development and application of molecular diagnostics in plant diseases. Molecular techniques used in plant disease diagnostics need to be more reliable, faster, and easier than conventional methods. Now the challenges are with scientists to develop practical techniques to be used for molecular diagnostics of plant diseases. Recent advancement in the improvement and application of molecular methods for diagnosing the widespread and emerging plant pathogenic fungi are discussed in this review.

Neurotuberculosis: an update.

Tuberculous (TB) meningitis (TBM), accounting for 70-80% of cases of neurotuberculosis, is one of the most severe forms of extrapulmonary tuberculosis. Two-thirds of new TB cases come from eight countries. Polymorphisms in toll-interleukin-1 receptor domain and in leukotriene A4 hydrolase (LTA4H) gene, affect the risk of inflammation in TBM. The common site of tuberculoma in children is cerebellum, and they may rarely develop tuberculous encephalopathy which has a high mortality. Young females with a high cerebrospinal fluid (CSF) protein have an increased predisposition to develop optochiasmatic arachnoiditis. Spinal TB meningitis may mimic transverse myelitis or Guillain-Barre syndrome. An extra-neural focus of TB should be sought clinically and radiologically as it may indicate safer and more accessible sites for diagnostic samplings. Cartridge-based nucleic acid amplification test (CBNAAT), also known as Genexpert test, is a polymerase chain reaction (PCR)-based method for detection of TB which also detects rifampicin resistance as it targets the rpob gene of mycobacteria. Line probe assays, based on PCR and reverse hybridization methods, identify mutations associated with drug resistance within a week. TBM being a paucibacillary disease, often evades a definite diagnosis and empirical treatment for a minimum of 9months is warranted based on clinical judgement. All TBM patients should receive adjunctive corticosteroids, even those with HIV infection. Drug resistance is strongly associated with previous treatment and bedaquiline as well as delamanid have received approvals for multidrug resistant (MDR) TB. The key principle of managing MDR TB is never to add a single drug to a failing regimen. Correct combination and duration of most effective second line drugs in MDR TB require further modifications. Early shunting should be considered in those with hydrocephalus failing medical management. The single most important determinant of outcome is the stage of TBM at which treatment has been started.

Improving peanut protein quality: Expression of a synthetic storage protein

Peanut is an affordable legume used in most households. It represents one of the most important protein supplies worldwide. However, peanut proteins are deficient in several essential amino acids (EAA), like most plant proteins; whereas plants are the main source of dietary proteins consumed by humans and livestock. This could lead to protein malnutrition in areas where people diet relies on one or two staple foods. Based on its high nutritional value, peanut is a good candidate for genetic biofortification. This study is aimed at expressing an EAA-rich artificial storage protein (ASPx) into peanut seeds for increased nutritive value. The ASPx derived gene was introduced into peanut via Agrobacterium-mediated transformation. Molecular analysis of regenerated kanamycin resistant plants using polymerase chain reaction (PCR) and Southern hybridization indicate the stable integration of one copy ASPx gene in transgenic plants. The expression of the ASPx in transgenic peanuts seeds was detected by mass spectrometry (multiple reaction monitoring). Amino acids analysis showed an increase of 12 to 19% of most EAA (Val, Tyr, Phe, Iso, Leu, Met) in a transgenic line. The results show that the nutritional quality of peanut could be improved. Key words: Genetic biofortification, essential amino acid, storage protein, peanut.

The PCR-HCR dual signal amplification strategy for ultrasensitive detection of Escherichia coli O157:H7 in milk

In this study, we established a novel polymerase chain reaction-hybridization chain reaction (PCR-HCR) dual signal amplification assay for sensitive detection of Escherichia coli O157:H7 in milk. In this assay, a blocker forward primer and a biotin reverse primer based on E. coli O157:H7 specific fliCh7 gene were specifically designed for PCR amplification resulting in a single-double stranded DNA with biotin (ss-dsDNA~biotin). These PCR products, ss-dsDNA~biotin, were captured by magnetic beads (MBs) decorated with streptavidin (SA) forming the ss-dsDNA~biotin+SA~MBs. The ssDNA on the PCR products opened the hairpin probe (H1 and H2) to trigger the HCR for secondary signal amplification. This is the first to use HCR to directly amplify PCR products through a novel blocker primer. With the help of two nucleic acid dual signal amplification techniques, PCR-HCR analysis can effectively improve the detection sensitivity and the limit of detection is 7.2 × 101 CFU/mL. In addition, this assay has been successfully used to detection target bacteria in skim milk spiked samples.

Incidence of betapapillomaviruses in the tumour and perilesional healthy skin in patients with basal cell carcinoma depending on sex, age, hair colour, tumour subtype, its location and dissemination.

IntroductionBasal cell carcinoma (BCC) is the most common skin cancer in the Caucasian population. It is believed that infections caused by viruses from the genus betapapillomavirus (β-HPV) might be associated with the risk of BCC, but the spread of data on the prevalence of the virus in biopsies is significant.AimTo assess the presence and diversity of β-HPV in skin samples taken from the tumour and a fragment of healthy skin from the patients with BCC, as well as checking the correlation of factors listed below and presence of β-HPV infection in the studied patients.Material and methodsThe study was conducted on the skin biopsies from 73 patients with histopathologically confirmed BCC. The following data were collected from patients: sex, age, hair colour and tumour location. Using the polymerase chain reaction (PCR) test, the presence of β-HPV infection was detected in the tested samples. PCR and reverse hybridization assay were also used to genotype 25 types of β-HPV.ResultsA statistically significant correlation was found between the sex and BCC type, BCC type and tumour location, BCC type and exposure to UV radiation, as well as between the hair colour and tumour location. The correlation between the BCC type and the number of tumours and HPV types detected was also noted.ConclusionsPreliminary studies suggest that one of the risk factors for development of infiltrating lesions is the presence of a single HPV 93 infection, but further research is needed to confirm these assumptions.

A rare case of miliary tuberculosis accompanying perihepatitis.

Perihepatic capsulitis is associated with various diseases, such as Fitz-Hugh-Curtis syndrome, systemic lupus erythematosus, perforated cholecystitis, perforated hepatic abscess, and tuberculous peritonitis. Miliary tuberculosis is present in about 2% of all reported cases of tuberculosis and is characterized by the widespread millet-like hematogenous dissemination of Mycobacterium tuberculosis. We describe a 24-year-old virgin patient presenting with right upper quadrant and costovertebral angle pain. Diffuse perihepatic capsular enhancement was observed in abdominal computed tomography scans. Chest radiography showed miliary tuberculosis, and a polymerase chain reaction hybridization assay of sputum revealed the presence of M. tuberculosis. Symptoms improved after administering anti-tuberculosis medications. This report describes a rare case of miliary tuberculosis accompanying perihepatitis.

SRNAPrimerDB: comprehensive primer design and search web service for small non-coding RNAs.

Small non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs), play key roles in many biological processes. However, only a few tools can be used to develop the optimal primer or probe design for the expression profile of small ncRNAs. Here, we developed sRNAPrimerDB, the first automated primer designing and query web service for small ncRNAs. The primer online designing module of sRNAPrimerDB is composed of primer design algorithms and quality evaluation of the polymerase chain reaction (PCR) primer. Five types of primers, namely, generic or specific reverse transcription primers, specific PCR primers pairs, TaqMan probe, double-hairpin probe and hybridization probe for different small ncRNA detection methods, can be designed and searched using this service. The quality of PCR primers is further evaluated using melting temperature, primer dimer, hairpin structure and specificity. Moreover, the sequence and size of each amplicon are also provided for the subsequent experiment verification. At present, 531306 and 2941669 primer pairs exist across 223 species for miRNAs and piRNAs, respectively, according to sRNAPrimerDB. Several primers designed by sRNAPrimerDB are further successfully validated by subsequent experiments. sRNAPrimerDB is a valuable platform that can be used to detect small ncRNAs. This module can be publicly accessible at http://www.srnaprimerdb.com or http://123.57.239.141. Supplementary data are available at Bioinformatics online.

Cervical small cell carcinoma frequently presented in multiple high risk HPV infection and often associated with other type of epithelial tumors

BackgroundSmall cell carcinoma of the uterine cervix is a rare and highly malignant tumor, and its etiopathogenesis is strongly related to high-risk HPV infections.MethodsThe clinicopathological data of 30 cases of cervical primary small cell carcinoma were retrospectively analyzed. In situ hybridization, polymerase chain reaction and reverse dot-blot hybridization were employed to detect HPV DNA in both small cell carcinoma and other coexisting epithelial tumors. Immunohistochemistry was used to detect the protein expression of p16 and p53.ResultsAmongst 30 patients with cervical primary small cell carcinoma, 15 patients simultaneously exhibited other types of epithelial tumors, including squamous cell carcinoma, adenocarcinoma, squamous cell carcinoma in situ, and adenocarcinoma in situ. Most tumor cells infected with HPV presented integrated patterns in the nuclei by in situ hybridization. HPV DNA was detected in every small cell carcinoma case (100%) by polymerase chain reaction and reverse dot blot hybridization. 27 cases (90%) harbored type 18, and 15 (50%) displayed multiple HPV18 and 16 infections. The prevalence of HPV 18 infection in small cell carcinoma was higher than in cervical squamous and glandular epithelial neoplasms (P = 0.002). However, similar infection rates of HPV 16 were detected in both tumors (P = 0.383). Both small cell carcinoma and other types of epithelial tumors exhibited strong nuclear and cytoplasmic staining for p16 in all cases. Three cases of small cell carcinoma revealed completely negative p53 immunohistochemical expression in 15 cases of composite tumors, which suggested TP53 nonsense mutation pattern. The pure small cell carcinoma of uterine cervix had similar mutation or wild type pattern for TP53 compared with composite tumor (P = 0.224).ConclusionsCervical small cell carcinomas are often associated with squamous or glandular epithelial tumors, which might result from multiple HPV infections, especially HPV 16 infection. Multiple HPV infections were not correlated with tumor stage, size, lymphovascular invasion, lymph node metastasis, or prognosis. Furthermore, careful observation of specimens is very important in finding little proportion of small cell carcinoma in the composite lesions, specifically in cervical biopsy specimens, in order to avoid the missed diagnosis of small cell carcinoma.

Analysis of the resistance of Gossypium herbaceum to cotton leaf curl kokhran virus strain burewala and cotton leaf curl multan betasatellite

Gossypium herbaceum is resistant to cotton leaf curl disease but the nature of the resistance is poorly characterized. The reaction of G. herbaceum cv. Co Tiep Khac to infection by the Burewala strain of cotton leaf curl Kokhran virus (CLCuKoV-Bur) and cotton leaf curl Multan betasatellite (CLCuMuB) was characterized following whitefly-mediated inoculation and grafting. The CLCuKoV-Bur and CLCuMuB isolates used in this study had 97–100% nucleotide sequence identity with other isolates for which sequence information is accessible in databases. A high level of resistance was demonstrated in cv. Co Tiep Khac using 200 whiteflies per plant with no apparent disease symptoms even 90 days post-inoculation, and no detection of the virus-betasatellite complex, as shown by polymerase chain reaction (PCR) and rolling circle amplification (RCA)/PCR. Following graft-inoculation, mild foliar disease symptoms were observed in leaves proximal to the grafting sites of most of cv. Co Tiep Khac plants but not in apical leaves. The presence of CLCuKoV-Bur and CLCuMuB was detected by RCA/PCR but not by PCR and Southern hybridization in symptomless apical leaves of cv. Co Tiep Khac. In addition, less virus accumulated in cv. Co Tiep Khac compared to G. hirsutum cv. CIM-496. These results provided insights into the resistance of G. herbaceum cv. Co Tiep Khac to CLCuKoV-Bur and CLCuMuB.

Rapid Detection of Ethambutol-Resistant Mycobacterium tuberculosis in Clinical Specimens by Real-Time Polymerase Chain Reaction Hybridisation Probe Method

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens. Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa. Materials and Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein–Jenson (L–J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB. Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L–J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L–J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing. Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.

Evaluation of the Performance of PCR and Hybrid Capture 2 in the Detection of Human Papillomavirus Associated with Cervical Cancer in Algeria

To evaluate and compare the performance of PCR (polymerase chain reaction) and second-generation capture hybridization (HC2) in the detection of high-risk Human Papillomavirus (HPV-HR) and to estimate their prevalence among normal cervical, dysplastic and cervical cancer in the Algerian population. 264 women were subjected to cyto-histological and colposcopic diagnostics and to the HPV test by PCR and Hybrid capture 2. The search for HPV DNA by conventional PCR was carried out with MY09 / 11 consensus primers and the typing of HPV with the hybridization technique in solution, Digene Sharp Signal for PCR. Hybrid Capture® 2 was performed with the digene HPV HC2 DNA Test kit. The PCR and HC2 performance tests included sensitivity, specificity, positive and negative predictive values, Youden's index, and Yule's Q coefficient. HPV-HR research has shown that these viruses are associated with 100% high-grade lesions, 88.2% by PCR and 83.3% by HC2 (p = 0.8) with invasive cancer. The PCR and HC2 performance tests were in favor of HCV HPV testing which was more sensitive, more specific, more predictive and more effective. The results on the prevalence of HPV-HR associated with cervical lesions are comparable to those in the literature. In comparison with PCR, the capture hybridization technique 2 is recommended for HPV diagnosis because of its better performance and simplicity.

Real-time PCR assay for the diagnosis of pleural tuberculosis

Introduction: The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak.Objective: To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis.Method: The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen or Mycobacterium tuberculosis culture in fluid or pleural tissue, or pleural biopsy with granulomas. Pleural tuberculosis was ruled out (in 31 patients) if all three tests were negative. The operating characteristics of the RT-PCR, using the Mid-P Exact Test, were determined using the OpenEpi 2.3 Software (2009).Results: The RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia.Conclusions: The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis.