Polymerase Chain Reaction Reaction Research Articles

Comparison of RNA Extraction and Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Detection of Porcine Reproductive and Respiratory Syndrome Virus in Porcine Oral Fluid Specimens

The objective of the current study was to evaluate various RNA extraction and polymerase chain reaction (PCR) protocols for the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) in porcine oral fluids. Extraction protocols were selected based on ease of use and compatibility with high-throughput, automated systems. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited by unknown factors in the oral fluid matrix and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids.

High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites-Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis-were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites.

Investigation of dogs as a reservoir of Penicillium marneffei in northern Thailand

Penicillium marneffei is a dimorphic pathogenic fungus endemic in Southeast Asia that usually causes disseminated disease, mainly in immunocompromised individuals, especially those with HIV infection. Untreated cases are usually fatal. The only known natural reservoir exists in bamboo rats and there is no firm evidence that these animals are involved in direct transmission to humans. The risk of infection is not restricted to those living in endemic areas; HIV-infected individuals who travel to Southeast Asia have also become infected by P. marneffei. Hence, there must exist sources to which even tourists are exposed on a short-term basis. Penicillium is known to infect dogs and this animal is common in the streets and temple areas of Chiang Mai, where there is one of the highest incidences of P. marneffei infection in the world. Dogs have not been well studied as a possible reservoir. To investigate this possibility, we took nasal swabs from 83 outdoor dogs and performed culture and nested polymerase chain reaction (PCR) to detect P. marneffei. We found that approximately 13% of nasal swabs from dogs in Chiang Mai, Thailand were positive when tested by two different PCR methods, but culture results were negative. Sequencing the products from both PCR reactions showed 100% identity with P. marneffei, whereas no other known fungi shared both sequences. Our results suggest that dogs might be an animal reservoir for P. marneffei in northern Thailand. This observation should be confirmed by additional studies.

Prevalence and relationship of human papilloma virus type 16 and type 18 with oral squamous cell carcinoma and oral leukoplakia in fresh scrappings: A PCR study

It has been always an area of diffuse clarity when you study malignancy and its pathogenesis. Recently, it has invited lot of interest among the researchers about the possibility of role of viruses in the initiation of carcinogenesis. Recent advances in the field of molecular biology and biotechnology have solved some problems with regard to pathogenesis. Human papilloma virus (HPV) and its role in the initiation of malignancy in the cervix is proven almost beyond doubt. The present study is aimed at the role of two types of HPV 16 and 18 in the initiation of oral premalignant and squamous cell carcinoma. The study also aims at using polymerase chain reaction (PCR) in finding out the prevalence of these types diagnosed histologically as oral leukoplakia and oral squamous cell carcinoma and prevalence of its association with the habit of tobacco use. In the present study, 45 patients having histopathologically confirmed oral squamous cell carcinoma in the age range of 32-85 years were selected along with 20 histopathologically confirmed oral leukoplakia in the age range 22-66 years. All the samples were subjected to polymerase chain reaction. The PCR reaction was carried out in PTC 200 thermo-cycler [MJ Research Inc, Watertown, MA, USA]. The site prevalence and co-infection rate of these two types of viruses are being analyzed using very simple non-invasive scrapings obtained from fresh scrapings and found to be really high. It was also observed that 73.3% (33/45) of the oral squamous cell carcinoma patients were positive for oral HPV type 16 while 71.1% (32/45) were positive for HPV type 18 infection and 57.7% (26/45) were found to have both HPV type 16 and HPV type 18 infections. HPV type 16, 18, and co-infection of both types showed high prevalence in oral squamous cell carcinoma.The prevalence of HPV type 18 was found to be higher than HPV type 16 and co-infection in oral leukoplakia. It was observed that the tongue and palate lesions in the oral squamous cell carcinoma patients showed high prevalence of HPV type 16, type 18, and co-infection compared with other sites.

Sensitive and specific identification by polymerase chain reaction ofEimeria tenellaandEimeria maxima, important protozoan pathogens in laboratory avian facilities

Eimeria tenella and Eimeria maxima are important pathogens causing intracellular protozoa infections in laboratory avian animals and are known to affect experimental results obtained from contaminated animals. This study aimed to find a fast, sensitive, and efficient protocol for the molecular identification of E. tenella and E. maxima in experimental samples using chickens as laboratory avian animals. DNA was extracted from fecal samples collected from chickens and polymerase chain reaction (PCR) analysis was employed to detect E. tenella and E. maxima from the extracted DNA. The target nucleic acid fragments were specifically amplified by PCR. Feces secreting E. tenella and E. maxima were detected by a positive PCR reaction. In this study, we were able to successfully detect E. tenella and E. maxima using the molecular diagnostic method of PCR. As such, we recommended PCR for monitoring E. tenella and E. maxima in laboratory avian facilities.

Identification of Herpesviruses Types 1 to 8 and Human Papillomavirus in Acute Apical Abscesses

Introduction It has been suggested that viruses, especially herpesviruses, can play a role in the pathogenesis of marginal and apical periodontitis. This study aimed to detect herpesviruses types 1 to 8, namely herpes simplex virus (HSV-1/2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), and human herpesvirus-8 (HHV-8) as well as human papillomavirus (HPV) in acute apical abscesses. Methods Twenty-four samples were taken by aspiration of the purulent exudate from acute apical abscesses. DNA extracted from clinical samples served as a template in single or nested polymerase chain reaction (PCR) assays for the detection of the target viruses. Results Control PCR reactions with β-globin gene primers revealed that all samples but one had detectable human DNA. Of the 23 abscess samples positive for the β-globin gene, 14 (61%) were positive for at least one of the target human viruses. Thirteen (56.5%) cases had herpesvirus: HHV-8 occurred in 11 (48%), VZV and HHV-6B in two (9%), and HHV-7 and HSV-1/2 in one (4%). EBV and HCMV were not present in any of the examined samples. HPV was detected in three (13%) abscess samples. Viral coinfection was found in five cases, with one case harboring three of the targeted viruses. Conclusion A large number of abscess samples were positive for at least one target virus. Unexpectedly, HHV-8 was for the first time detected and in a high prevalence. Papillomavirus and other herpesviruses were also found for the first time in endodontic abscesses. Although these findings suggest an association, the specific role of viruses in the pathogenesis of acute apical abscesses awaits further clarification.

Development of the nine X-STR loci typing system and genetic analysis in three nationality populations from China

This study is to develop a new multiplex polymerase chain reaction (PCR) system that simultaneously amplifies the nine X-chromosome short tandem repeats loci in the same PCR reaction, and to explore their polymorphism and mutation rate among three nationality populations from China. These loci included DXS6854, DXS9902, DXS6809, GATA172D05, HPRTB, DXS7423, DXS6807, DXS8378, and DXS8377. The samples of 890 (484 males and 406 females) unrelated individuals from Guangdong Han population, Xinjiang Uigur, and Inner-Mongolia Mongol were successfully analyzed using this multiplex system. The allele frequencies and mutation rates of the nine loci were investigated, and the comparison of allele frequency distribution among different populations was performed. There were 87 alleles for all the loci, and six to 18 alleles for each locus observed by our new multiplex PCR system. Polymorphism information content was 0.4998-0.9101, and power of discrimination in females was 0.6518-0.9846. Five cases with mutation of above loci were detected in 5,310 meioses. Pair-wise comparisons of allele frequencies distribution showed significant differences for most loci among different populations. Our results indicate that this multiplex system is very useful for identification analysis, and that the information about polymorphism and mutation rate is necessary for forensic application in three nationality populations from China.

Application of cDNA Macroarray for Simultaneous Detection of 12 Potato Viruses.

A complementary DNA (cDNA) macroarray was developed for simultaneous detection of 12 different potato viruses. A suitable region in the viral genome for each was selected for Alfalfa mosaic virus, Cucumber mosaic virus, Potato aucuba mosaic virus, Potato leafroll virus, Potato mop-top virus, Potato virus A, Potato virus M, Potato virus S, Potato virus X, Potato virus Y, Tomato ringspot virus, and Tomato spotted wilt virus, and their respective cDNAs were cloned into plasmid vectors. Capture probes for each virus ranging from 290 to 577 bp were generated by polymerase chain reaction (PCR) and immobilized on a nylon membrane. Total RNAs were extracted from each of these virus infected-plants, and cDNAs were synthesized from the RNA extracts using a random 9-mer primer. Subsequently, PCR reactions were performed using one primer pair for each of the 12 viruses. During PCR, amplified cDNAs were labeled with biotin and used as a target for hybridization analyses on a macroarray membrane. Hybridization signals between capture probes for the 12 viruses and their respective target cDNAs were observed using chemiluminescent or colorimetric detection. In all viruses, hybridization signals with capture probes were detected only when homologous virus targets were examined, and no hybridization to healthy plant extract was observed, facilitating identification of each virus. The results by colorimetric detection agreed with those obtained using chemiluminescence. The macroarray method developed was 5 × 102 to 4 × 106 times more sensitive than enzyme-linked immunosorbent assay and 5 to 5 × 104 times more sensitive than reverse-transcription PCR, except for Alfalfa mosaic virus. Colorimetric detection and substantial reduction in cross-hybridization signals much improved the method compared with other array-based detection methods for practical use.

Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ) are linked to hibernating state in hamsters

BackgroundThe structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR) is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR) tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB) and hibernating (HIB) hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators.ResultsBoth autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p < 0.01) prevalence of α1 ratio (over total α subunits considered in the present study) in the medial preoptic area (MPOA) and arcuate nucleus (Arc) of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%). Regarding the other two subunits (β and γ), elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions.ConclusionWe conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional distribution pattern of distinct GABAAR subunit combinations may prove to be very useful for highlighting GABAergic mechanisms functioning at least during the different physiological states of hibernators and this may have interesting therapeutic bearings on neurological sleeping disorders.

Molecular Detection of Autosomal-Dominant Feline Polycystic Kidney Disease by Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction

Feline autosomal-dominant polycystic kidney disease (ADPKD), with its characteristic growth of fluid-filled cysts of different sizes, is the most prevalent inherited genetic disease of cats. The point mutation (C-->A transversion) in exon 29 of the PKD1 gene is known to contribute to ADPKD development and can thus serve as a target for the molecular genetic diagnosis of ADPKD. To this end, a simple amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) was designed with 3 primers: 2 forward primers specifically targeting either the mutant or normal allele, and 1 universal reverse primer for amplification of both alleles. The new method was tested on the DNA from 35 feline blood samples, which included 15 mutant cats and 20 wild type cats. As verified by direct DNA sequencing, both sensitivity and specificity of this tri-primer ARMS PCR were 100%. As the multiplex ARMS PCR test can be performed in a single PCR reaction without other post-PCR procedures, it is a simple and accurate method for molecular studies of feline ADPKD.

Ovarian stimulation retards postimplantation development and alters global gene expression profile of blastocysts in mouse

This study compares the quality, developmental capacity and global gene expression profile of embryos generated from superovulated and natural cycles. We found that ovarian stimulation impaired quality of blastocysts (cell number: 68.9 +/- 6.9 vs. 76.8 +/- 7.9; diameter of blastocysts: 145.3 +/- 76.9 microm vs. 152.6 +/- 65.5 microm), fetus development (rate of development to term: 45.5% vs. 69.1%; weight of 18.5-dpc fetuses: 1.23 +/- 0.03 g vs. 1.34 +/- 0.03 g) and caused 92 genes differentially expressed.

Quantitative assay for the detection of the V617F variant in the Janus kinase 2 (JAK2) gene using the Luminex xMAP technology

BackgroundThe availability of clinically valid biomarkers contribute to improve the diagnosis and clinical management of diseases. A valine-to-phenylalanine substitution at position 617 (V617F) in the Janus kinase 2 (JAK2) gene has been recently associated with key signaling abnormalities in the transduction of haemopoietic growth-factor receptors and is now considered as a useful clinical marker of myeloproliferative neoplasms. Several methods have recently been reported to detect the JAK2 V617F point mutation and show variable sensitivity.MethodsUsing the Luminex xMAP technology, we developed a quantitative assay to detect the JAK2V617F variant. The method was based on polymerase chain reaction (PCR) followed by hybridization to specific probes coupled with internally dyed microspheres. The assay comprises 3 steps: genomic DNA extraction, end point PCR reaction, direct hybridization of PCR fragments and quantification. It has been tested with different sources of nucleic acid.ResultsApplied to whole blood samples, this quantitative assay showed a limit of detection of 2%. A highly sensitive allele-specific primer extension reaction performed in parallel allowed to validate the results and to identify the specimens with values below 2%.ConclusionDirect hybridization assay using the Luminex xMAP technology allows sensitive quantification of JAK2V617F from blood spots. It is simple and can be easily performed in a clinical setting.

Design of improved polymerase chain reaction (PCR) method containing internal positive control (IPC) for molecular detection of Yersinia pestis

Background: Plague is one of the three epidemic diseases still subject to the international health regulation and notifiable to the WHO. Yersinia pestis, the causative agent of plague in natural foci transmitted between rodent and human via wild rodent fleas. So set up of suitable and sure to surveillance and prevention of the disease is necessary. The molecular methods such as polymerase chaine reaction (PCR) has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC) to monitor for false negative results. Therefore we improved a uniplex and multiplex PCR method with lower of limit of detection. Methods: PCR reactions performed with primers which targeted of the caf1 and pla genes located on the pFra and pPst plasmids and the irp2 chromosomal gene located on the ‘pathogenicity island. For acquired different size of these genes aditional primers were designed. In each gene two parts required were ligated.The limit of detection determined by performing PCR reactions on serial dilutions of plasmids containing the coresponding inserts.for evaluating the specificity, PCR reactions were done for negative control bacteria. Results: Assays were performed with genome of Y. pestis which produced three DNA fragments of the expected size 300, 400 and 520 base pairs (bp) corresponding to irp2, caf1 and pla genes respectively. also, for IPC by the same primers the different fragment of the expected size 150, 500 and 300 base pairs (bp) corresponding to irp2, caf1 and pla genes respectively were acquired. With The lower limit of detection was 370 copy numbers for caf1 gene and 21 for pla gene. In PCR reactions for negative control bacteria detectable fragments were not observed. Conclusion: Our method clearly discriminated Y. pestis DNA bacteria which were tested. The rapidity, specificity and sensitivity of this procedure with the use of IPC to monitor for false negative results can make this method suitable for diagnosis and suggest that it can serve as a useful alternative method for inoculation of laboratory animals or the use of specific culture media for routine plaque surveillance and outbreak investigations. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive

Comparison of BIOMED-2 Versus Laboratory-Developed Polymerase Chain Reaction Assays for Detecting T-Cell Receptor-γ Gene Rearrangements

Detecting clonal T-cell receptor (TCR)-gamma gene rearrangements (GRs) is an important adjunct test for diagnosing T-cell lymphoma. We compared a recently described assay (BIOMED-2 protocol), which targets multiple variable (V) gene segments in two polymerase chain reaction (PCR) reactions (multi-V), with a frequently referenced assay that targets a single V gene segment in four separate PCR reactions (mono-V). A total of 144 consecutive clinical DNA samples were prospectively tested for T-cell clonality by PCR using laboratory-developed mono-V and commercial multi-V primer sets for TCR-gamma GR. The combination of TCR-beta, mono-V TCR-gamma and multi-V TCR-gamma detected more clonal cases (68/144, 47%) than any individual PCR assay. We detected clonal TCR-beta GR in 47/68 (69%) cases. Using either mono-V or multi-V TCR-gamma primers, the sensitivities for detecting clonality were 52/68 (76%) or 51/68 (75%). Using both mono-V and multi-V TCR-gamma primers improved the sensitivity for detecting clonality, 60/68 (88%). Combining either mono-V or multi-V TCR-gamma primers with TCR-beta primers also improved the sensitivity, 64/68 (94%). Significantly, TCR-gamma V11 GRs could only be detected using the mono-V-PCR primers. We conclude that using more than one T-cell PCR assay can enhance the overall sensitivity for detecting T-cell clonality.

Isolation, characterization and evaluation of significant mycoflora and mycotoxins in pig feed from Serbian farms

This paper provides a brief review of approaches for the early detection and prevention strategies which have been employed in Serbia for the control of ochratoxogenic fungi and its metabolites in feed in the context of a hazard analysis critical control point (HACCP) framework. During a mycological analysis of complete feedmixes intended for fattening swine (n = 18), a total of six genera and 14 species of moulds were identified. Penicillium was present in considerably more samples than any other genus (94.4%), followed by the genera Fusarium (55.5%) and Paecilomyces (44.4%). Other fungi from the genera Aspergillus (22%), Mucor (11.1%) and Alternaria (5.5%) were represented in a smaller amount. Total fungal counts ranged from 105 to 40 × 105 c.f.u./g. The mycotoxins deoxynivalenol, ochratoxin A and zearalenone were detected, while aflatoxins were not present. Deoxynivalenol was detected in 10 samples in the concentration range 0.25–2.5 mg/kg. Ochratoxin A and zaralenone were detected in nine and eight samples, respectively, in the concentration range 0.057–0.27 and 0.2–5.0 mg/kg, respectively. Isolates identified as Aspergillus and Penicillium species were subjected to molecular characterization for the presence of genes responsible for the synthesis of OTA (polyketide synthase gene-PKS) using polymerase chain reaction (PCR) applied to a set of 18 isolates. The sequences of PCR reaction products in three samples were compared with nucleotide sequences of genes for polyketide synthase (PKS) from Penicillium species and it was found that the samples possessed the PKS sequence. These findings indicate that there may be a risk of animal exposure to mycotoxins through the consumption of mouldy infected feeds.

Conventional and molecular diagnostic testing for the acute neurologic patient.

Objective – The aim of this review is to describe and evaluate both conventional and molecular diagnostic testing utilized in dogs and cats with acute neurologic diseases. Various types of polymerase chain reaction (PCR) are explored along with novel molecular diagnostic testing that ultimately may prove useful in the critical care setting. Data Sources – PUBMED was searched to obtain relevant references material using keywords: ‘canine OR feline meningitis AND meningoencephalitis,’‘feline infectious peritonitis,’‘canine distemper,’‘canine OR feline AND toxoplasma,’‘canine neospora,’‘canine OR feline AND rickettsia,’‘granulomatous meningoencephalitis,’‘steroid responsive meningitis arteritis,’‘necrotizing encephalitis,’‘novel neurodiagnostics,’‘canine OR feline AND CNS borrelia,’‘canine OR feline AND CNS bartonella,’‘canine OR feline AND CNS fungal,’‘nested OR multiplex OR degenerate OR consensus OR CODEHOP AND PCR.’ Research findings from the authors' laboratory and current veterinary textbooks also were utilized. Human Data Synthesis – Molecular diagnostic testing including conventional, real‐time, and consensus and degenerate PCR and microarray analysis are utilized routinely for the antemortem diagnosis of infectious meningoencephalitis (ME) in humans. Recently, PCR using consensus degenerate hybrid primers (CODEHOP) has been used to identify and characterize a number of novel human viruses. Veterinary Data Synthesis – Molecular diagnostic testing such as conventional and real‐time PCR aid in the diagnosis of several important central nervous system infectious agents including canine distemper virus, Toxoplasma gondii, Neospora caninum, rickettsial species, and others. Recently, broadly reactive consensus and degenerate PCR reactions have been applied to canine ME including assays for rickettsial organisms, Borrelia spp. and Bartonella spp., and various viral families. Conclusions – In the acute neurologic patient, there are several key infectious diseases that can be pursued by a combination of conventional and molecular diagnostic testing. It is important that the clinician understands the utility, as well as the limitations, of the various neurodiagnostic tests that are available.

Comparative efficacy of conventional primer sets in detection of Cryptosporidium parvum for diagnostic use

In this study, the sensitivity and specificity of different previously described primer sets for Cryptosporidium parvum detection by polymerase chain reaction (PCR) was evaluated. For this purpose, the primer sets defined by Cacciò et al. (FEMS Microbiol Lett 170(1):173-179, 1999) (tub), Widmer et al. (Appl Environ Microbiol 64(11):4477-4481, 1998) (btub) and Rochelle et al. (Appl Environ Microbiol 63:2029-2037, 1997) (cphsp), respectively, were used. Deoxyribonucleic acid (DNA) was isolated from three different sample materials: (1) from the faeces of an experimentally C. parvum-infected calf, (2) from purified C. parvum oocysts, and (3) from C. parvum-infected HCT-8 cell cultures. The DNA samples were subjected to PCR reactions with each of the three given primer sets to investigate sensitivity and suitability for routine use. The primers described by Cacciò et al. (FEMS Microbiol Lett 170(1):173-179, 1999) (TUB) were superior regarding sensitivity and specificity in terms of detection of C. parvum in faeces, in purified oocysts and also in cell culture, and may thus be applied for routine diagnostic use in common sample materials.

Distribution of angiotensin II receptor expression in the microcirculation of striated muscle.

The current study was undertaken to localize and identify angiotensin II (Ang II) receptor subtypes in the microcirculation of striated muscle. Cremaster muscles from 7- to 8-week-old Sprague-Dawley rats were excised, placed in a dissection solution maintained at 4 degrees C, and 3 branch orders of arterioles and venules, as well as capillaries and a muscle specimen, were microdissected under a stereomicroscope. Reverse transcription polymerase chain reaction (RT-PCR) methods were developed for purification and amplification of extremely small amounts of RNA (<5 ng/microL) from whole tissue samples. RNA was isolated from each sample, reverse transcribed, and the cDNA products were amplified by polymerase chain reactions (PCR) specifically primed with either AT(1a), AT(1b), or AT(2) receptor primers. The products were electrophoretically size-fractionated on an agarose gel, stained with ethidium bromide to visualize DNA bands, and analyzed to determine the presence or absence of AT receptor subtypes. Protein expression was confirmed by Western blotting pooled samples with specific antiserum. AT(1a) and AT(2) receptors were found in nearly all orders of both arterioles and venules, as well as the skeletal muscle biopsies. AT(1b) receptors, if present, were only observed on a few instances in the arterioles. Furthermore, PCR reactions specifically primed for skeletal muscle cell (MHC(2B)) and endothelial cell (eNOS) specific proteins demonstrated that there was no cross-contamination between the vessels and the skeletal muscle biopsies. This study describes a unique method for the isolation and preparation of microvessels and provides the first data directly demonstrating the presence of AT(1) and AT(2) receptors in microvessels as well as in skeletal muscle fibers.

Constraints to estimating the prevalence of trypanosome infections in East African zebu cattle

BackgroundIn East Africa, animal trypanosomiasis is caused by many tsetse transmitted protozoan parasites including Trypanosoma vivax, T. congolense and subspecies of T. brucei s.l. (T. b. brucei and zoonotic human infective T. b. rhodesiense) that may co-circulate in domestic and wild animals. Accurate species-specific prevalence measurements of these parasites in animal populations are complicated by mixed infections of trypanosomes within individual hosts, low parasite densities and difficulties in conducting field studies. Many Polymerase Chain Reaction (PCR) based diagnostic tools are available to characterise and quantify infection in animals. These are important for assessing the contribution of infections in animal reservoirs and the risk posed to humans from zoonotic trypanosome species. New matrices for DNA capture have simplified large scale field PCR analyses but few studies have examined the impact of these techniques on prevalence estimations.ResultsThe Whatman FTA matrix has been evaluated using a random sample of 35 village zebu cattle from a population naturally exposed to trypanosome infection. Using a generic trypanosome-specific PCR, prevalence was systematically evaluated. Multiple PCR samples taken from single FTA cards demonstrated that a single punch from an FTA card is not sufficient to confirm the infectivity status of an individual animal as parasite DNA is unevenly distributed across the card. At low parasite densities in the host, this stochastic sampling effect results in underestimation of prevalence based on single punch PCR testing. Repeated testing increased the estimated prevalence of all Trypanosoma spp. from 9.7% to 86%. Using repeat testing, a very high prevalence of pathogenic trypanosomes was detected in these local village cattle: T. brucei (34.3%), T. congolense (42.9%) and T. vivax (22.9%).ConclusionsThese results show that, despite the convenience of Whatman FTA cards and specific PCR based detection tools, the chronically low parasitaemias in indigenous African zebu cattle make it difficult to establish true prevalence. Although this study specifically applies to FTA cards, a similar effect would be experienced with other approaches using blood samples containing low parasite densities. For example, using blood film microscopy or PCR detection from liquid samples where the probability of detecting a parasite or DNA molecule, in the required number of fields of view or PCR reaction, is less than one.

ELIMINATION OF OXIDATIVE COMPOUNDS TO INCREASE THE QUALITY AND QUANTITY OF DNA EXTRACTED FROM LEAVES OF GUAVA (PSIDIUM GUAJAVA L.)

With the aim of extracting DNA in sufficient quantity and quality from dry leaves of guava preserved for about a year, three different methods were evaluated. Only with one of them DNA was obtained, however, it was necessary to make some modifications to eliminate products derived from oxidative processes, oxidation promoting compounds, as well as pigments that are particularly abundant in the guava leaves and even more when they are preserved for a long period of time at room temperature. The DNA obtained was used to carry out the Polymerase Chain Reaction (PCR). To verify its concentration, measurements were made by spectrophotometry and the quality was verified by electrophoresis in agarose gels as well as by different tests with PCR amplifications. Also, the method was used with fresh leaves and DNA with good characteristics was obtained to conduct the PCR reactions. In addition, this method will allow making the collecting of plant material easier in the field because cryo-preservation agents are not required for the transportation of plant material.