Nucleic Acid Hybridization Techniques Research Articles

History of advances in genetic engineering of viruses before COVID-19 pandemic.

On December 31, 2019, the World Health Organization's China Country Office was alerted to cases of pneumonia of unknown cause detected in Wuhan City, Hubei Province of China. Due to the fact that to date, the question of the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been resolved yet, the author analyzed the main advances in the development of genetic engineering of viruses that took place before the onset of the COVID-19 pandemic. The first artificial genetically modified viruses could appear in nature in the mid-1950s. The technique of nucleic acid hybridization was developed by the end-1960s. In the late 1970s, a method called the "reverse genetics" emerged to synthesize ribonucleic acid and deoxyribonucleic acid molecules. In the early 1980-s, it became possible to combine the genes of different viruses and insert the genes of one virus into the genome of another virus. Since that time, the production of vector vaccines began. At present, by modern technologies one can assemble any virus based on the nucleotide sequence available in the virus database or designed by a computer as a virtual model. Scientists around the world are invited to answer the call of Neil Harrison and Jeffrey Sachs of Columbia University, for a thorough and independent investigation into the origin of SARS-CoV-2. Only a full understanding of the origin of the new virus can minimize the likelihood of a similar pandemic in the future.

STUDY OF THE ROLE OF RNA-INTERFERENCE IN THE REGULATION OF DISCRETE FRAGMENTATION OF 18S RRNA IN PLANTS

Under stressful conditions, cells must rapidly reduce the level of total mRNA translation, while maintaining the synthesis of some special proteins that help resist stress. In animals and yeast, the inhibition of protein synthesis is mainly associated with the modification of the translation initiation and elongation factors. In plant cells, many of these regulatory mechanisms are not realized. We hypothesize that plants have an alternative mechanism for suppressing mRNA translation associated with site-specific fragmentation of 18S rRNA molecules within 40S ribosomal subunits. Understanding the molecular mechanisms of plant response to stress is the basis for increasing the efficiency of the breeding process in order to develop stress-resistant varieties of economically important plant species. We found that during a heat shock, discrete RNA molecules accumulate in the cells of various plant species, which are 5'-terminal fragments of 18S rRNA that remain bound in the composition of ribosomal subunits. In this work, the method of nucleic acid hybridization was used to study RNA-interference as a possible mechanism mediating the phenomenon of discrete fragmentation of 18S rRNA in plants in response to heat shock. MicroRNA molecules complementary to the target regions of 18S rRNA, through which its rupture was carried out with the formation of stress-induced 5'-terminal RNA fragments, were not found. The small 5'-terminal fragments of 18S rRNA revealed during the work are proposed to be used as a new type of stress biomarkers in plant cells. The optimized nucleic acid hybridization technique using a DIG-labeled probe complementary to the highly conserved 1-35 5'-end region of plant 18S rRNA can be used to efficiently detect these stress RNA markers.

Prevalence and genetic diversity of Trichomonas vaginalis in the general population of Granada and co-infections with Gardnerella vaginalis and Candida species.

Purulent or exudative genitourinary infections are a frequent cause of consultation in primary and specialized healthcare. The objectives of this study were: to determine the prevalence of Trichomonas vaginalis and co-infections with Candida spp. and Gardnerella vaginalis in vaginal secretion; and to use multilocus sequence typing (MLST) to analyse the genetic diversity of T. vaginalis strains. The samples were submitted for analysis (n=5230) to a third-level hospital in Granada (Southern Spain) between 2011 and 2014; eight T. vaginalis strains isolated during 2015 were randomly selected for MLST analysis. Culture and nucleic acid hybridization techniques were used to detect microorganisms in the samples. The prevalence of T. vaginalis was 2.4 % between 2011 and 2014, being higher during the first few months of both 2011 and 2012. Among samples positive for T. vaginalis, co-infection with G. vaginalis was detected in 29 samples and co-infection with Candida spp. in 6, while co-infection with all three pathogens was observed in 3 samples. The only statistically significant between-year difference in co-infection rates was observed for T. vaginalis with G. vaginalis due to an elevated rate in 2011. MLST analysis results demonstrated a high genetic variability among strains circulating in our setting. These findings emphasize the need for the routine application of diagnostic procedures to avoid the spread of this sexually transmitted infection.

The relationship between human papillomavirus genotypes and cervical lesions

Objective To investigate the prevalence and genotype distribution of human papilloma virus (HPV) infection among patients with cervical carcinoma or precancerous lesions in Xuzhou region. Methods 688 patients with cervical lesions confirmed by colposcopic biopsy were assigned to three groups: low-grade squamous intraepithelial lesion (LSIL) (187 cases), high-grade squamous intraepithelial lesion (HSIL) (359 cases) and cervical carcinoma (142 cases). The genotypes of HPV were determined by nucleic acid hybridization technique. The prevalence and genotype distribution of HPV were further analyzed. Results The infection rates of HPV in LSIL , HSIL and ICC group were 68.98 % (129/187), 94.99 % (341/359), 95.78 % (136/142), respectively. These rates were statistically significant different (P< 0.05). With the aggravation of cervical lesions, HR-HPV infection rate was gradually increased and LR-HPV infection rate was gradually decreased. The most common HPV types were listed below in order of decreasing prevalence. LSIL: HPV16, 52, 58, 18, 31; HSIL: HPV16, 58, 33, 31, 52; cervical carcinoma: HPV16, 31, 58, 18, 33. Based on the cumulative logistic regression model, confidence intervals of the relevance between the overall HPV prevalence and occurrence of cervical lesions were calculated: HPV16 (OR = 7.215, P< 0.05), HPV31 (OR = 2.768, P< 0.05), HPV33 (OR = 1.846, P< 0.05), HPV58 (OR = 1.726, P< 0.05) Conclusions The prevalence of HR-HPV infections increased with the aggravation of cervical carcinoma in Xuzhou region. In order of decreasing prevalence, the most prevalent of HPV types are HPV16, 31, 33, 58. The results indicate that HPV18 have strong tumorigenicity, but is not popular in Xuzhou region. By contrast HPV52 is more common in LSIL than that in cervical carcinoma. Key words: Human papillomavirus; Genotypes; Uterine cervical diseases

Use of Molecular Markers to Improve the Agro-Industrial Productivity in the North West of Argentina

Genetic improvement of crop plants through conventional breeding has been the cornerstone to improve global agricultural production. Nowadays, different molecular biology techniques have become available to supplement and assist conventional breeding, among them the utilization of molecular markers. The generation of molecular, or DNA, markers are based on two basic molecular biology methods in order to be able to detect polymorphism: Southern blotting, a nucleic acid hybridization technique, and the amplification in vitro of specific DNA segments by the Polymerase Chain Reaction (PCR) technique . By using these two methods and several variations of the basic technologies, many different kind of molecular markers have been developed during the last decades. It is important to note that for a molecular marker to be efficient in crop breeding it should meet most of the following criteria [: high level of polymorphism; even distribution across the whole genome (not clustered in certain regions); co-dominance (or capacity to identify at heterozygous); clear distinct allelic features; single copy; low cost to use (or cost-efficient marker development and genotyping); easy assay/detection and automation; high availability (un-restricted use) and suitability to be duplicated/multiplexed (so that the data can be accumulated and shared between laboratories); genome-specific in nature (especially with polyploids) and have no detrimental effect on phenotype. However, the choice of the best marker system depends on many other factors as well including crop genetics and available resources (time, reagents, equipments, etc).

Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species

The development and evaluation of a protocol based on polymerase chain reaction (PCR) and nucleic acid hybridization techniques for the specific detection of lymphocystis disease virus (LCDV) in several marine fish species. The pair of primers for PCR, OBL3 and OBL4, was designed based on published nucleotide sequence (LCDV-1) and amplifies a fragment within the major capsid protein. The sensitivity was evaluated using DNA from purified viral particles, as well as from cells inoculated with several viral concentrations. The PCR combined with slot blot was the most sensitive methodology, detecting 2.5 ng of viral DNA. Using this methodology LCDV was detected at 5 days postinoculation from SAF-1 cells initially inoculated with 10(-5) TCID(50) ml(-1). The combination of PCR with membrane hybridization has also been proved to be adequate to detect LCDV from apparently healthy carriers by means of caudal fin sample analysis. This asymptomatic infection was also demonstrated by classical virological methods (cell culture and immunoblot). The protocol described in this study allows the specific detection of LCDV, both in cell cultures and in fin homogenates from asymptomatic fish. The detection of asymptomatic carriers by a rapid molecular method using caudal fin sampling, which does not imply animal killing, could be an important tool to control epizootics caused by LCDV, as fish could be analysed before their introduction and/or mobilization in farm facilities.

ISOLATION AND IDENTIFICATION OF Spiroplasma citri USING SOME SENSITIVE METHODS

Spiroplasma citri, the causal agent of stubborn disease, was isolated from leaves and fruits of diseased citrus plants and cultured on solid and liquid C3G medium. On the basis of mode of transmission, symptomatology, shape on solid medium, staining of infected plant tissues with Dienes’ stain and phase contrast microscopy, the isolated agent was identified as Spiroplasma citri. Identifiction was ensured by ELISA and PCR techniques. A fragment (1053 bp) from the spiralin gene of S. citri was amplified by PCR using two specific primers for the spiralin gene. The nucleic acid hybridization techniques (Southern and dot blots) were used for identification of the spiroplasmal genome using non-radioactive DNA probe specific for spiralin gene region. It was carried out as an alternative sensitive method for rapid detection of the Egyption isolate of S. citri using non-radioactive DNA probe specific for spiralin gene region.

OCCULT PLASMODIUM VIVAX INFECTION DIAGNOSED BY A POLYMERASE CHAIN REACTION–BASED DETECTION SYSTEM: A CASE REPORT

After a trip to Zambia, a previously healthy adult traveler presented with a prolonged illness characterized by low-grade fevers and fatigue. Although malaria smears and antibody tests results for Plasmodium species were negative, a diagnosis of malaria was ultimately determined by polymerase chain reaction (PCR) amplification and species-specific nucleic acid hybridization techniques. The patient was successfully treated and cured. Clinical use of PCR technology may facilitate the identification of cases of smear-negative malaria, which up to the present time, have been difficult to diagnose.

Induced CYP1A mRNA, protein and catalytic activity in the liver of feral fish, leaping mullet, Liza saliens

In this study, we examined whether levels of P4501A mRNA expression were naturally induced in feral fish, Liza saliens, and whether CYP1A protein levels and associated enzyme activity, EROD, were also increased. Induction of mRNA was measured using a nucleic acid hybridization technique. For the hybridization studies, a new 33-mer oligonucleotide probe 5′-dCTC ATC CAG CTT CCT GTC CTC GCA GTG ATC AAT-3′ was designed, which corresponded to the totally conserved amino acid motif of CYP1A protein from positions 291 to 301 among the various fish species. Results of Northern blot analysis revealed that RNA isolated from the liver of mullet collected from the highly contaminated region of Izmir Bay with a dissolved and dispersed petroleum hydrocarbon content of 12.45 μg l −1 gave a strong hybridization signal, whereas only a weak hybridization signal was detected in the liver RNA of fish caught from the reference site containing less than 1 μg l −1 of petroleum hydrocarbons. Similarly, fish from the contaminated site had approximately 80 times more EROD activity than the feral fish captured from the reference site. Studies using polyclonal antibodies produced against purified mullet CYP1A also showed the similar trend. In conclusion, feral leaping mullet caught from contaminated water displayed induction of CYP1A at three levels of expression, namely, mRNA, apoprotein and catalytic activity.

Molecular Techniques for Determining Microbial Diversity and Community Structure in Natural Environments

The ability to quantify the number and kinds of microorganisms within a community is fundamental to the understanding of the structure and function of an ecosystem. The simple morphology of most microbes provides few clues for their identification and physiological traits are often ambiguous. In addition, many organisms resist cultivation, which is essential to their characterization. Recombinant DNA techniques have provided a means whereby many of the obstacles associated with cultivation and description can be overcome and subsequently has allowed many new insights into the complexity of natural microbial communities. Molecular approaches based on 16S ribosomal RNA (rRNA) sequence analysis allow direct investigation of the community structure, diversity, and phylogeny of microorganisms in almost any environment, while quantification of the individual types of microorganisms or entire microbial communities may be addressed by nucleic acid hybridization techniques. Furthermore, the use of fluorescently labeled population-specific rRNA probes allows microscopic examination of individual cells in complex microbial assemblages as well as their interactions in situ. In this review, we discuss strategies for characterizing microbial communities without the need for cultivation.

Diagnosis and treatment of cytomegalovirus infection.

Cytomegalovirus (CMV) infection remains a major cause of morbidity and mortality in recipients of an allogeneic stem cell transplant (SCT). Due to the broad application of antiviral prophylaxis and preemptive therapy, a decrease in early-onset and a subsequent increase in late-onset CMV disease has been observed. New data on the latency state and reactivation of CMV have been presented, the role of T-cell responses in the control of CMV further substantiated, and viral immune escape mechanisms described in more detail. Sensitive diagnostic assays using nucleic acid amplification and hybridization techniques have been commercialized and will allow standardization of CMV diagnostics in antiviral drug trials. Quantification of the viral load will be increasingly considered for initiation and, in patients with persistence of high viral titers despite antiviral therapy, screening for antiviral drug resistance. Clinical data are emerging to show that, apart from ganciclovir, foscarnet can be given safely even after allogeneic SCT. Additional drugs like lobucavir and cidofovir have been used for specific indications. Interactions of immunosuppressive drugs and antiviral compounds of clinical relevance have been described. Thus, therapeutic drug monitoring and targeted antiviral drug dosing will become standard practice in antiviral treatment strategies in patients following allogeneic SCT.

MOLECULAR CHARACTERIZATION OF THE HUMAN INTERLEUKIN (Il)-17 RECEPTOR

Human interleukin 17 (hIL-17) is a T-cell derived cytokine that exhibits 63% amino acid sequence identity to mouse IL-17 (mIL-17) and 57% identity to a viral protein encoded by the herpesvirus saimiri (HSV) gene 13 (HVS13). The IL-17 family of proteins binds to a unique mouse receptor (mIL-17R). Using nucleic acid hybridization techniques, a cDNA encoding a human homologue of the mIL-17R (hIL-17R) was isolated from a human T cell library. The predicted amino acid sequence of the hIL-17R is 69% identical to the mIL-17R, shares no homology with previously identified cytokine receptor families, and exhibits a broad tissue distribution. The hIL-17R gene was localized to chromosome 22. Monoclonal antibodies (mAbs) generated against the hIL-17R were able to block the IL-17-induced production of cytokine from human foreskin fibroblast (HFF) cells. Binding studies suggest that recombinant hIL-17 binds to the hIL-17R with low affinity.

Comparison of Techniques for Detection of Barley Yellow Dwarf Virus-PAV-IL.

Detection of barley yellow dwarf virus (BYDV)-PAV-IL by an improved nucleic acid hybridization technique, using a nonradioactive probe with chromogenic and chemiluminescent substrates, was compared with detection by polymerase chain reaction (PCR), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antibodies, and triple antibody sandwich ELISA with polyclonal and monoclonal antibodies. Each method was used to detect purified virus and virus in sap extracts from infected oat leaves. The detection limits for both ELISA procedures were 1 ng of purified BYDV-PAV-IL and the equivalent of 78 ng of infected tissue. Nucleic acid hybridization with either chemiluminescent or chromogenic substrates also detected as little as 1 ng of purified BYDV-PAV-IL, but it was slightly more sensitive at detecting virus in tissue extracts (25 ng of infected tissue). The most sensitive detection technique was PCR amplification, which could detect as little as 0.1 pg of RNA extracted from purified virus and detected viral RNA in the equivalent of 0.5 pg of infected leaf tissue.

Neisseria gonorrhoeae RNA/DNA hybridization and culture for screening of gonococcal infections in a low-prevalence population.

Gonorrhea is still a major sexually transmitted disease (STD) worldwide. Its etiologic diagnosis is based on identification of the causative agent, Neisseria gonorrhoeae, by culture of genital secretions, which is often hampered by difficulties of sample collection and transport. Alternatively, nucleic acid hybridization techniques for routine diagnosis of N. gonorrhoeae appear to be useful by eliminating problems associated with bacterial viability, particularly for surveillance of low-prevalence populations. Our study among 1,508 outpatients undergoing routine examination for common STDs used RNA/DNA hybridization with a DNA probe specific for N. gonorrhoeae (Gen Probe Pace 2) and classical culture. Of the 1,750 specimens tested, 12 were positive by DNA probe and culture. In 8 cases, only DNA probe was positive while culture was negative. In 3 of these discrepant cases clinical and epidemiological data suggested true N. gonorrhoeae infection. Thus, DNA probe assay for N. gonorrhoeae may greatly improve screening of N. gonorrhoeae among low-prevalence populations. However, culture remains mandatory for testing antimicrobial resistance of these highly communicable infectious agents.

Enzymatic labeling of nucleic acids.

Enzymatic labeling of nucleic acids is a fundamental tool in molecular biology with virtually every aspect of nucleic acid hybridization technique involving the use of labeled probes. Different methods for enzymatic labeling of DNA, RNA and oligonucleotide probes are available today. In this review, we will describe both radioactive and nonradioactive labeling methods, yet the choice of system for labeling the probe depends on the application under study.

La evolución de técnicas y fenómenos: hacia una explicación de la "confección" del mundo

In recent articles some authors (e.g. Pickering 1989, Hacking 1992) have pointed out a process of gradual adjustment or tailoring between phenomena, models and experimental techniques. However, the whole idea of tailoring or adjusting has been dealt with as a mere metaphore. In this paper we present an evolutionary model of phenomena and techniques which explains this gradual adjustment or tailoring as an adaptative causal process, i.e. not as a mere metaphor. Our aim is accomplished in three steps. First, we arrive at the general conditions that changes in a population of entities with reproductive capabilities have to satisfy in order to be modelled as an evolutionary process, in a causal-explanatory sense. We show that a characterization of the class of experimental techniques (a class associated with an experimental tradition) meet these conditions, and we examine in detail how the nucleic acid hybridization techniques used in molecular biology can be modelled in the way we propose. A second step is to show that the sort of variability that metters in evolutionary models of techniques and phenomena is aggregative variability, i.e. the sort of variability that can be selected. This is an important point, since most evolutionary models of technical and scientific change in the literature fail to satisfy this requirement. A common objection to evolutionary models of scientific change is that fitness, the central notion of evolutionary models in population biology, has no counterpart in these models. We show that our model can provide a natural concept of fitness, a concept that has a similar role to play in our model as in biological models. Finally, as a third step, we conclude with an explanation of how the world can be said to be tailored. It is the result of an evolutionary process which incorporates inextricably related conceptual and material resources. In this sense, the world consists of phenomena that are made by us, but which are not mere inventions of our mind.

Specific activation in jun-transformed avian fibroblasts of a gene (bkj) related to the avian beta-keratin gene family.

We have analyzed differential gene expression in normal versus jun-transformed avian fibroblasts by using subtracted nucleic acid probes and differential nucleic acid hybridization techniques for the isolation of cDNA clones. One clone corresponded to a gene that was strongly expressed in a previously established quail (Coturnix japonica) embryo fibroblast line (VCD) transformed by a chimeric jun oncogene but whose expression was undetectable in normal quail embryo fibroblasts. Furthermore, the gene was expressed in quail or chicken fibroblast cultures that were freshly transformed by retroviral constructs carrying various viral or cellular jun alleles and in chicken fibroblasts transformed by the avian retrovirus ASV17 carrying the original viral v-jun allele. However, its expression was undetectable in a variety of established avian cell lines or freshly prepared avian fibroblast cultures transformed by other oncogenes or a chemical carcinogen. The nucleotide and deduced amino acid sequences of the cDNA clone were not identical to any sequence entries in the data bases but revealed significant similarities to avian beta-keratin genes; the highest degree of amino acid sequence identity was 63%. The gene, which we termed bkj, may represent a direct or indirect target for jun function.

A review on the diagnosis infection in cattle of Schistosoma bovis: current status and future prospects

Bovine schistosomiasis, caused by Schistosoma bovis, is a serious veterinary problem in many parts of the worid. The current methods used for the diagnosis of the disease include clinical signs, pathological lesions, parasitological and serological techniques. As clinical signs and parasitological lesions caused by S. bovis are indistinguishable from those induced by other trematode parasites, confirmation of diagnosis by these methods is unreliable. Parasitological techniques used to demonstrate eggs of the parasite in fecal or tissue samples represent the most accurate method for detection of an active S. bovis infection. The tissue of choice for detection of S. bovis infection is the liver because of the visible macroscopic lesion that can be seen in that organ and the rapid detection of the parasite eggs under the microscope using crush smears. The serological techniques used for diagnosis of the disease do not necessarily identify an active infection. In addition, some of the positive reactions are non specific. However, serology is useful to identify previous infection in epidemiologic study. The ELISA has been recentiy validated for the diagnosis of bovine schistosomiasis and will probably replace the other serological tests. The immunoblotting technique has been proven satisfactory to detect antibodies to defined and recombinant schistosome antigen vaccines. Nucleic acid hybridization techniques have been described for the study of schistosome species-specific identification. However, these molecular techniques have not yet revolutionarized diagnosis of schistosomiasis. These techniques will probably serve as the basis for future diagnostic tests.

Characterization of the diversity of sulfate-reducing bacteria in soil and mining waste water environments by nucleic acid hybridization techniques.

Nucleic acid hybridization techniques were used to characterize the sulfate-reducing bacterial communities at seven waste water and two soil sites in Canada. Genomic DNA was obtained from liquid enrichment cultures of samples taken from these nine sites. The liquid enrichment protocol favored growth of the sulfate-reducing bacterial component of the communities at these sites. The genomic DNA preparations were analyzed with (i) a specific gene probe aimed at a single genus (Desulfovibrio), (ii) a general 16S rRNA gene probe aimed at all genera of sulfate-reducing bacteria and other bacteria, and (iii) whole genome probes aimed at specific bacteria. This three-pronged approach provided information on the sulfate-reducing bacterial community structures for the nine sites. These were compared with each other and with the sulfate-reducing bacterial communities of western Canadian oil field production waters, studied previously. It was found that there is considerable diversity in the sulfate-reducing bacterial community at each site. Most sulfate-reducing bacteria isolated from distinct sites are genomically different and differ also from sulfate-reducing bacteria found in oil field production waters.

Chemiluminescent detection of potato spindle tuber viroid in true potato seed using a digoxigenin labelled DNA probe

The potential use of a simple, sensitive and non-radioactive method for detecting potato spindle tuber viroid (PSTVd) in germinated true potato seeds, based on nucleic acid hybridization with a PSTVd-specific DNA probe labelled with digoxigenin, was investigated. Two simple procedures for the clarification of plant extracts suitable for a non-radioactive detection system were also investigated. The nucleic acid hybridization technique could detect one PSTVd-infected seed in more than 150 healthy seeds. The benefits of this non-radioactive detection system are discussed.