Nucleic Acid Hybridization Methods Research Articles

Effects of 1,8-cineole on neuropathic pain mediated by P2X2 receptor in the spinal cord dorsal horn

As an intractable health threat, neuropathic pain is now a key problem in clinical therapy, which can be caused by lesions affecting the peripheral nervous systems. 1,8-cineole is a natural monoterpene cyclic ether present in eucalyptus and has been reported to exhibit anti-inflammatory and antioxidant effects. Research has shown that 1,8-cineole inhibits P2X3 receptor-mediated neuropathic pains in dorsal root ganglion. The P2X2 and P2X3 receptors participate in the transmission of algesia and nociception information by primary sensory neurons. In the present study, We thus investigated in the spinal cord dorsal horn whether 1,8-cineole inhibits the expression of P2X2 receptor-mediated neuropathic pain. This study used rats in five random groups: group of chronic constriction injury(CCI) with dimethysulfoxide control (CCI + DMSO); group of CCI; sham group(Sham); group of CCI treated with a low dose 1,8-cineole (CCI + 50 mg/kg); group of CCI with a high dose (CCI + 100 mg/kg). We observed the effects of 1,8-cineole on thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT). We examined P2X2 receptors mRNA change in rat spinal cord dorsal horn by In situ nucleic acid hybridization(ISH) and Quantitative realtime polymerase chain reaction (qRT-PCR) methods. Western Blotting and Immunohistochemical staining methods were used to observe P2X2 receptor protein expressions in the rat spinal cord dorsal horn. It demonstrated that oral administration of 1,8-cineole inhibits over-expression of P2X2 receptor protein and mRNA in the spinal cord and dorsal horn in the CCI rats. And the study explored new methods for the prevention and treatment of neuropathic pain.

Detection and quantification of Parascaris P-glycoprotein drug transporter expression with a novel mRNA hybridization technique

Macrocyclic lactone-resistant Parascaris have been reported throughout the world. In part, the drug resistant phenotype is hypothesized to be associated with ATP-binding cassette transporters known as P-glycoproteins. In many systems, P-glycoproteins efflux drugs out of cells thereby precluding drug binding to target receptors. Parascaris may evade macrocyclic lactone-mediated death by effluxing drugs away from target receptors in the nervous system. Alternatively, P-glycoprotein expression in the gut or body wall could prevent penetration of drugs into the body of the parasite altogether. In the present study, we evaluate expression of Peq-pgp-11 and Peq-pgp-16 using a novel multiple nucleic acid hybridization method. This method allowed for visualization of individual mRNA transcripts within fixed tissue sections of Parascaris adults. Our investigation revealed expression of Peq-pgp-11 and Peq-pgp-16 in the intestine, body wall, nerves, lateral cords, and reproductive tissues of male and female parasites. These results suggest that Pgp could efflux drugs locally at the level of parasite neuronal tissue as well as at sites of entry for drugs such as the hypodermis and intestine. The multiple nucleic acid hybridization method could be useful for providing tissue context for gene expression in a variety of nematode parasites.

Detection of Common Bacterial Causes of STIs among STD Clinic Attendees by Gram Stain and Culture versus the Microarray Nucleic Acid Hybridization Method

Objectives: The purpose of the study was to determine the prevalence and etiology of predominant sexually transmitted infections (STIs) occurring among the STD and Gynecology Clinic attendees and to compare sensitivity and specificity of conventional versus molecular diagnostic methods. Methods: A total of 1,441 individuals were recruited from the STD clinic at the National Center for Communicable Diseases and Gynecology Clinic at the National Center for Oncology between 2014 and 2015. Urogenital tract swab samples were collected and tested for N.gonorrhoeae, C.trachomatis, and T.vaginalis using conventional (Gram stain and culture) and molecular (Microarray Nucleic Acid Hybridization - MNAHM) diagnostic methods. The final infection status of participants was determined using the “either test positive” strategy. Results: Of the total 1,441 participants, 19.7% were N.gonorrhoeae infected, 12.5% were C.trachomatis infected, and 7.8% were T.vaginalis infected. For T. vaginalis, conventional methods and MNAHM had sensitivities of 17.9% and 98.2%, with specificities of 96.6% and 99.6%, respectively. For N.gonorrhoeae, conventional methods had sensitivity and specificity values of 33.3% and 99.1%, and MNAHM had values of 95.1% and 98.8%, respectively. Conclusion: The study found a high prevalence of N.gonorrhoeae and C.trachomatis among STD and Gynecology Clinic attendees. Conventional methods for detection of N.gonorrhoeae and T.vaginalis were significantly less sensitive than molecular diagnostic methods and didn’t detect other STI pathogens.

High Expression of Neutrophil Elastase Predicts Improved Survival In Pediatric Acute Myeloid Leukemia: A Report From the Children's Oncology Group

AbstractAbstract 2762 Background:Recent studies indicated that high expression of neutrophil elastase, a primary granule serine protease encoded by ELANE (ELA2), predicts longer survival of patients with chronic-phase chronic myeloid leukemia (CML) and improved outcome following allogeneic hematopoietic stem cell transplantation for advanced-phase CML. In contrast, the prognostic role of neutrophil elastase in acute myeloid leukemia (AML) is unknown. Therefore, we evaluated its expression in participants in a recent Children's Oncology Group (COG) AML pilot protocol. Methods:COG-AAML03P1 enrolled 340 newly diagnosed children (aged 1 month to 21 years) with de novo AML, excluding those with acute promyelocytic leukemia and Down syndrome, and tested the feasibility of combining gemtuzumab ozogamicin (GO) with intensive induction chemotherapy followed by GO-containing intensification therapy or matched related donor stem cell transplantation. Diagnostic marrow specimens from 113 randomly selected patients were used for ELANE mRNA quantification using a sandwich nucleic acid hybridization method employing branched DNA molecules to amplify the signal from captured target RNA. Expression levels were normalized using beta glucuronidase (GUSB) and then correlated with patient demographics, laboratory characteristics, and clinical outcome. Cytogenetics and molecular prognostic markers were used for risk classification as follows: low risk (mutation in core-binding factor, NPM1, or CEBPa; n=43), high risk (−5/5q−, monosomy 7, or FLT3-ITD with high allelic ratio; n=16), or standard risk (all other patients with cytogenetic/molecular data; n=54). Results:Among the 113 diagnostic specimens, ELANE mRNA expression levels were undetectable in 3 samples and varied more than 70,000-fold relative to GUSB in the rest, from 0.0005 to 36.32 (median, 0.89). Patients with high ELANE expression (>median expression) were more likely to have myelomonocytic cell characteristics (FAB M4, p<0.001) and more likely to have core-binding factor translocations, t(8;21) (p=0.05) or inv(16) (p<0.003). They had a lower prevalence of megakaryocytic leukemia (p<0.02). There was no difference in the prevalence of FLT3-ITD or NPM1 mutations. As a result of these characteristics, patients with high ELANE expression were more likely to have low-risk disease (p<0.001) at the expense of standard-risk disease, while the prevalence of high-risk disease was similar. We subsequently determined the clinical outcome per median ELANE expression for the entire cohort as well as the specific risk groups. In the entire cohort, patients with high ELANE expression had a significantly superior 3-year overall survival (OS; 80±11% vs 61±13%, p=0.045) and tended to have better disease-free survival (66±14% vs 51±16%, p=0.118). Subgroup analyses were limited by the small sample size. Nevertheless, in both high- and low-risk disease, patients with high ELANE expression tended to have a better 3-year OS than patients with low ELANE expression (67±38% vs 30±29%, p=0.308 for high risk; 90±11% vs 75±25%, p=0.361 for low risk). By comparison, OS in standard-risk patients appeared similar for patients with high and low ELANE expression (70±21% vs 66±17%, p=0.849). In multivariate Cox regression analyses that included age, cytogenetics, and molecular prognostic factors, high ELANE expression was associated with reduced overall mortality (hazard ratio: 0.66); however, this result did not reach statistical significance (95% confidence interval: 0.33–1.35, p=0.260), possibly because of the small sample size. Conclusion:This study revealed significant heterogeneity of ELANE expression in pediatric AML and its association with improved OS. This association is at least partly due to the high proportion of AMLs with core-binding factor aberrations in patients with high ELANE expression. Nevertheless, high ELANE expression may predict improved OS even within specific risk groups, in particular high-risk disease, although further studies with a larger patient cohort will be necessary to confirm this finding. Disclosures:No relevant conflicts of interest to declare.

Splicing of a Bacterial Group II Intron from Bacillus megaterium Is Independent of Intron-Encoded Protein

The splicing of a bacterial group II subclass B intron B.me.I1 from Bacillus megaterium chromosomes was investigated. RT-PCR and nucleic acid hybridization methods were used to understand the role of the intron-encoded protein (IEP) in the splicing of B.me.I1. An in vivo assay showed that the splicing occurred in the absence of IEP. An in vitro assay showed that B.me.I1 was spliced under conditions similar to those of the intracellular environment with no help from other biological molecules. Because all group II introns previously reported needed IEPs for their splicing in vivo, our results suggest that B.me.I1 is an "actual" self-splicing group II intron. This is also the first report to recognize the existence of group II introns that independently splice mRNA in vivo. The self-splicing of a bacterial intron may support that eukaryotic spliceosomal introns originated in bacterial genomes.

Diagnosis of Coccidioidomycosis by Culture

The recovery of Coccidioides spp. by culture and confirmation utilizing the AccuProbe nucleic acid hybridization method by GenProbe remain the definitive diagnostic method. Biosafety considerations from specimen collection through culture confirmation in the mycology laboratory are critical, as acquisition of coccidioidomycosis by laboratory workers is well documented. The designation of Coccidioides spp. as select agents of potential bioterrorism has mandated strict regulation of their transport and inventory. The genus appears generally susceptible, in vitro, although no defined breakpoints exist. Susceptibility testing may assist in documenting treatment failures.

Detection of Group A beta-hemolytic Streptococcus employing three different detection methods: culture, rapid antigen detecting test, and molecular assay.

In order to study the prevalence of Group A beta-hemolytic Streptococcus (GABHS) pharyngotonsillitis in our pediatric population and to compare different sampling methods of GABHS detection, oropharyngeal swabs from 50 children with acute pharyngotonsillitis, between 1 and 12 years old, were used simultaneously for culture, molecular assay and rapid GABHS antigen detection tests. All children were clinically examined at the Division of Pediatric Otorhinolaryngology of the Federal University of São Paulo. Diagnostic criteria were based on signs and symptoms, including sore throat, fever and oropharyngeal purulent secretion. Children that had been treated with antibiotics were excluded. Overall, combining the three methods, the prevalence of GABHS was 34%. GABHS was diagnosed in 30% of the bacterial cultures, in 25% of the samples tested with the molecular nucleic acid hybridization method and in 26% of the cases tested with the rapid antigen detection test. There was no significant difference between these three methods.

Rapid and accurate identification of mycobacteria by sequencing hypervariable regions of the 16S ribosomal RNA gene.

We developed a method to identify mycobacteria by sequencing hypervariable regions of the polymerase chain reaction-amplified 16S ribosomal RNA gene. This method is nearly specific for mycobacteria and uses positive culture from liquid or solid medium without the needfor lengthy subculture. It shortens identification time to 3 days, which is much faster than the conventional biochemical method (mean, 8 weeks). It applies to all mycobacteria (approximately 100 species), unlike current nucleic acid hybridization methods, which probe only 4 species. The identifications are the same or are species specific for the well-characterized mycobacteria (59/68 [87%]) or more accurate for recently proposed species (9/68 [13%]). The method requires a single sequencing reaction, which is efficient and cost-effective. Therefore, this method is clinically and academically useful.

Chrysanthemum stunt pospiviroid

Chrysanthemum stunt pospiviroid

High-throughput expression profiling techniques.

Great optimism exists that correlating genomics with function will lead to a better understanding of the detailed workings of the nervous system. The nearly complete sequencing of the genomes of several eukaryotic species and the invention of high-throughput expression profiling techniques now provide the means to rapidly investigate the molecular underpinnings of phenotypic change with reasonable accuracy. This approach is beginning to have an impact in neurobiology and we can expect it to be similarly useful for investigating problems in the chemical senses. Careful thought is necessary, however, in selecting and matching specific techniques and tissues, in confirming differences in mRNA abundance and in interpreting the results. A symposium on Functional Genomics in Neural Systems was held during the AChemS XXIII meeting to illustrate both the potential and the limitations of these techniques for investigating questions of importance to neurobiology in general and to the chemical senses in particular. A brief technical introduction, summarized herein, was followed by two keynote talks. Dr Daniel Geschwind (UCLA) shared his work identifying genes whose expression is correlated with differentiating neurons in developing neural tissues. Moving then to the other end of a nerve cell’s life, Dr Tomas Prolla (University of Wisconsin) described different sets of genes that are affected in the aged brain and how many of the age-related changes can be prevented by restricting the intake of calories. Both speakers incorporated their experiences and perspectives on the technical challenges that are a critical element in the successful use of these new and still improving techniques. Expression profiling techniques allow the simultaneous analysis of the abundance of many thousands of transcripts. By doing so, they provide three advantages. (i) They accelerate the discovery of transcripts whose abundance is correlated with any particular phenotype. (ii) Even more exciting than discoveries about individual transcripts, however, is the ability to observe patterns that emerge from comparing the known functions of all the affected transcripts. (iii) Finally, these techniques can provide hypothesis testing about gene expression patterns underlying phenotypic change on a broad scale. Clearly, expression profiling techniques provide powerful tools. The tools available for expression profiling can be grouped into three types (Table 1). (i) Clone and count methods generate thousands of 9–20 bp sequences representing specific sites near the 3′ ends of poly(A+) RNAs (Velculescu et al., 1995; Brenner et al., 2000). Sequences of this length are sufficient to uniquely identify most transcripts. The abundance of a transcript is measured simply as the number of times its sequence tag is encountered. Overall, this is a highly effective method, but laborious and expensive enough that it is less common than the other two types. (ii) Microarray methods depend upon hybridization of probes derived from RNA samples against DNA or RNA spots bound to a solid substrate (Schena et al., 1995). The commercial production of microarrays, the familiarity of investigators with nucleic acid hybridization methods and the rapidity of data collection have made this the most popular type of expression profiling method. Its limitations include the obvious fact that it is restricted to analyzing only those sequences present on the array and that it is slightly less sensitive than the other methods. The paper by Prolla (Prolla, 2002) that follows describes the details of expression profiling with Affymetrix GeneChip oligonucleotide arrays. Numerous reviews describing the details of, and differences among, microarray methods are available (Duggan et al., 1999; Lipshutz et al., 1999; Hegde et al., 2000; Luo and Geschwind, 2001). (iii) Differential subtraction methods use the power of PCR, adapting its conditions to favor amplification of differentially abundant cDNAs. The first invented was differential display (Liang and Pardee, 1992). More recently devised techniques, such as representational difference analysis (RDA) of cDNA (Hubank and Schatz, 1994) and suppressive subtractive hybridization (Diatchenko et al., 1996), share its advantages and appear to be more robust and accurate. For example, RDA of cDNA rarely yields false positives, is sequence-independent, interrogates the majority of transcripts simultaneously and can be used with very small amounts of tissue. Not that it lacks limitations, however. Analysis of the products is laborintensive and its dynamic range can be limited when many differences are present (though this can be at least partially overcome by repeating the procedure). For all three types of expression profiling methods there exists one major drawback—achieving the number of repetitions required for standard statistical analyses is often impossible or im-

Screening for Chlamydia trachomatis in asymptomatic women in Hungary. An epidemiological and cost-effectiveness analysis.

A multicenter survey was carried out in order to determine the prevalence and risk factors for Chlamydia trachomatis infection in the population of asymptomatic women in Hungary. Results were used to carry out a cost-effectiveness analysis of screening for chlamydial infection in women with asymptomatic genital infections. The non-amplified nucleic acid hybridization method (PACE 2 Gen-Probe) was used to diagnose C. trachomatis and Bayes' theorem was applied to assess the prevalence of the infection. Multiple logistic regression analysis was performed to differentiate the risk factors for chlamydial infections. According to the test, the prevalence of Chlamydia trachomatis among 1300 pregnant women was 4.5%. The sensitivity and specificity of the test are estimated to be 70% and 99%, respectively. After Bayes' correction, the overall estimated prevalence of chlamydial infection was 5.1%. There were significant differences in proportions of chlamydial infection in different regions, and also in different age groups and different family status groups. The highest rate was for women aged below 20 years: 16.9%. Cost-effectiveness analysis, with associated sensitivity analysis was carried out for women aged below 20 years. Three screening strategies were compared: using the ELISA method, using amplified Gen-Probe method and no screening. The amplified Gen-Probe method was best provided, the infection prevalence exceeded 16.7%, the PID rate exceeded 24% and the probability of tubal infertility in untreated women exceeded 25%. We conclude that screening with amplified Gen-Probe assays (followed by treatment of positive patients) is the preferred screening strategy for young women in Hungary.

Chlamydia trachomatis infection and the risk of perinatal mortality in Hungary.

Chlamydial infections of the genital tract are thought to often lead to preterm birth, which is the most important perinatal problem in Hungary. A multicenter study was carried out to determine the prevalence of Chlamydia trachomatis infection, risk factors for the infection and to relate the infection to perinatal mortality, accounting for potential confounding effects. The nucleic acid hybridization method (PACE2 Gen-Probe) was applied for the examination of Chlamydia trachomatis. Logistic regression analysis was used to assess risk. A total of 6156 pregnancy women were examined for the occurrence of Chlamydia trachomatis. The observed overall rate of chlamydial infection was 5.9%. Young age (less than 24 years old) (OR and 95% CI: 1.6 (1.3-2.0)), unmarried status (1.5 (1.2-1.9)) and the high unemployment rate (2.1 (1.6-2.7)) were statistically significant predictors of the infection. In logistic regression analysis, chlamydial infection (1.9 (1.1-3.3)), high unemployment rate (1.5 (1.2-2.2)) and low birth weight (1.7 (1.1-2.7) were significant predictors of perinatal mortality. Testing pregnant women for diseases that can be transmitted perinatally is an important part of obstetric care. Screening for C. trachomatis of unmarried women under 24 years of age is suggested and need increased observation during labor.

Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore

A previously presented homogeneous assay method, named the excimer-forming two-probe nucleic acid hybridization (ETPH) method, is based on specific excimer formation between two pyrenes attached at the neighboring terminals of two sequential probe oligonucleotides complementary to a single target. In this study, we investigated assay conditions and optimal molecular design of probes for intense excimer emission using a pyrenemethyliodoacetamide-introduced 16mer probe, a pyrene butanoic acid-introduced 16merprobe and a target 32mer. The length of the linker between the pyrene residue and the terminal sugar moiety remarkably influenced the quantum efficiency of excimer emission; the pair of linker arms of these two probes was optimal. The quantum efficiency was also dependent upon the concentrations of dimethylformamide and NaCl added to the assay solution. Spectroscopic measurements and T m analysis showed that an optimal configuration of the two pyrene residues for intense excimer emission might be affected by pyrene-pyrene interaction, pyrene-duplex interaction (intercalation/stacking) and solvent conditions as a whole. We then demonstrated the practicality of the ETPH method with the optimal hybridization conditions thus attained by determining that the concentration of 16S rRNA in extracts from Vibrio mimicus ATCC 33655 cells in exponential growth phase is 18 500 16S rRNA molecules/cell on average.

Prevalence of Chlamydia trachomatis infection in a low-risk population in Hungary.

Chlamydia trachomatis is the leading cause of nongonococcal urethritis and cervicitis in women. Because of the recent increases in the numbers of new cases and severe consequences, there is an urgent demand for the introduction of sensitive and specific rapid diagnostic methods. A multicenter examination involving seven centers was sponsored by the Hungarian Ministry of Health and Welfare in order to provide a survey of Chlamydia trachomatis in the gravid population. 6,161 women were tested between 1994 to 1995. The seven centers were selected with regard to different aspects, from developed and less developed areas in the capital, two large provincial towns, and various other provincial regions reflecting either an industrial or an agricultural background. The nucleic acid hybridization method (PACE 2 Gen-Probe, San Diego, CA) was introduced in this low-risk population for the examination of Chlamydia trachomatis. In one center, a further two methods, antigen detection by ELISA (SYVA, CA) and cultivation on the McCoy cell line (staining with SYVA FITC-labeled antichlamydia monoclonal antibody), were applied. International surveys and experience indicate that the proportion of the population threatened by Chlamydia trachomatis is above 10%. The overall average incidence of Chlamydia trachomatis cases in this low-risk gravid population was 5.74%. The data from the different centers ranged between 1.6% and 9.7%. The chlamydia-infected Hungarian gravid population is below the critical 10%, but there is one Hungarian county where the value is close to 10%. In this provincial, industrial area, the number of unmarried and divorced gravida in a low economic situation is disproportionately high. For this disadvantaged population, permanent Chlamydia trachomatis screening was suggested. In the other centers, screening of pregnant women for Chlamydia trachomatis and the treatment of positive cases and their partners were suggested for pathological gravida with preterm labor and preterm rupture of the membranes.

5597911 Mycobacterial nucleic acid hybridization probes and methods of use : Guesdon Jean-Luc; Thierry Dominique; Ullmann Agngra eGicquel Brigitt; Brisson-Noel Anne Paris, France Assigned to Institut Pasteur

5597911 Mycobacterial nucleic acid hybridization probes and methods of use : Guesdon Jean-Luc; Thierry Dominique; Ullmann Agngra eGicquel Brigitt; Brisson-Noel Anne Paris, France Assigned to Institut Pasteur

In situ hybridization studies of cytomegalovirus and Epstein-Barr virus in reactive histiocytic hyperplasia with hemophagocytosis.

We studied 14 adult patients presenting with fever and cytopenia of the peripheral blood and histiocytic hyperplasia with hemophagocytosis (HHH) in the bone marrow regarding an association of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) by using in situ hybridization (ISH) and also evaluated the clinical and laboratory findings according to the encountered organisms. ISH using a CMV RNA probe demonstrated infected cells in 6 out of 14 cases (43%), and ISH using an EBV EBER RNA probe demonstrated infected nuclei in 5 out of the same 14 cases (36%) of HHH. No cases showed a positive reaction with both probes. Three cases showed a negative reaction with both probes. The mean age of all patients was 29 years; and that of the CMV-positive patients was 27 years and that of the EBV-positive patients was 36 years. Organomegaly was found in 3 out of 6 CMV-positive patients (1 hepatomegaly, 1 splenomegaly, 1 hepatosplenomegaly), and 4 out of 5 EBV-positive patients (lymphadenopathy in all 4 cases, hepatosplenomegaly in 2 cases). One of the CMV-positive case had acute myeloblastic leukemia, and 2 EBV-positive cases had underlying malignancy (1 Hodgkin's disease, 1 non-Hodgkin's lymphoma). Seven out of the 14 HHH cases (50%) died within several months after diagnosis. Nucleic acid hybridization methods can be used for the routine examination of the association of CMV or EBV.

5403711 Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is : Walder Joseph; Walder Roxanne Y Iowa City, IA, United States Assigned to University of Iowa Research Foundation

5403711 Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is : Walder Joseph; Walder Roxanne Y Iowa City, IA, United States Assigned to University of Iowa Research Foundation

5403711 Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved : Walder Joseph; Walder Roxanne Y Iowa City, IA, United States Assigned to University of Iowa Research Foundation

5403711 Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved : Walder Joseph; Walder Roxanne Y Iowa City, IA, United States Assigned to University of Iowa Research Foundation

Nucleic acid hybridization accompanied with excimer formation from two pyrene-labeled probes.

We developed a novel nucleic acid hybridization method based on excimer formation. We used two different 16-mer oligonucleotide probes that had a combined continuous-sequence run that was complementary to a target 32-mer. Prior to hybridization, the adjacent terminal ends (i.e. the 3'-terminal of one probe and the 5'-terminal of the other probe) were each labeled with one pyrene residue. When these probes simultaneously hybridized to the target, a 495 nm broad fluorescence band was produced. The intensity of this band increased as the intensity of the pyrene monomer bands decreased, indicating that the 495 nm band was attributed to the pyrene excimer. The excimer fluorescence, easily differentiated from the monomer bands for emission wavelength, opens up a new way to perform homogeneous hybridization assays and in vivo imaging of nucleic acids.

Identification of Bacillus strains isolated from milk and cream with classical and nucleic acid hybridization methods.

A total of 529 bacterial strains have been isolated from milk and cream sampled at different sites in a dairy production plant under conditions selective for aerobic sporeforming bacteria. Identification with classical methods based on morphological, physiological and biochemical criteria showed Bacillus licheniformis to be the most frequently occurring Bacillus sp. The investigation also revealed 62 unidentified strains. Classical identification methods were time consuming (3-7 d), lacked specificity and--because of their dependence on phenotypic gene expression--sometimes produced ambiguous results. Consequently, a colony hybridization method developed for the identification of B. licheniformis strains and using nonradioactive labelled 23S rRNA targeted oligonucleotide probes was also used. Identification of B. licheniformis with both classical and hybridization methods revealed diverging identification results for 70 strains.