Recent brazilian experience on farmer reaction and crop response to fertilizer use

(1) In the period 1965/77 fertilizer consumption in Brazil increased nearly fifteen foild from circa 200,000 tons of N + P2O5 + K2O to 3 million tons. During the fifteen years extending from 1950 to 1964 usage of the primary macronutrients was raised by a factor of 2 only. (2) Several explanations are given for the remarkable increase, namely: an experimental background which supplied data for recommendations of rates, time and type of application; a convenient governmental policy for minimum prices and rural credit; capacity of the industry to meet the demand of the fertilizer market; an adequate mechanism for the diffusion of the practice of fertilizer use to the farmer. (3) The extension work, which has caused a permanent change in the aptitude towards fertilization, was carried out in the traditional way by salesmen supported by a technical staff, as well as by agronomists of the official services. (4) Two new programs were started and conducted in a rather short time, both putting emphasis on the relatively new technology of fertilizer use. (5) The first program, conducted in the Southern part of the country, extended lab and green house work supplemented by a few field trials to small land owners - the so called "operação tatú" (operation armadillo). (6) The seconde program, covering a larger problem area in the Northeast and in Central Brazil, began directly in field as thousands of demonstrations and simple experiments with the participation of local people whose involvement was essential for the success of the initiative; in this case the official extension services, both foreign and national sources of funds, and universities did participate under the leadership of the Brazilian Association for the Diffusion of Fertilizers (ANDA). (7) It is felt that the Brazilian experience gained thereof could be useful to other countries under similar conditions.

A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

Hepatitis C prevalence and risk factors in hemodialysis patients in Central Brazil: a survey by polymerase chain reaction and serological methods

An hemodialysis population in Central Brazil was screened by polymerase chain reaction (PCR) and serological methods to assess the prevalence of hepatitis C virus (HCV) infection and to investigate associated risk factors. All hemodialysis patients (n=428) were interviewed in eight dialysis units in Goiânia city. Blood samples were collected and serum samples screened for anti-HCV antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive samples were retested for confirmation with a line immunoassay (LIA). All samples were also tested for HCV RNA by the PCR. An overall prevalence of 46.7% (CI 95%: 42-51.5) was found, ranging from 20.7% (CI 95%: 8.8-38.1) to 90.4% (CI 95%: 79.9-96.4) depending on the dialysis unit. Of the 428 patients, 185 were found to be seropositive by ELISA, and 167 were confirmed positive by LIA, resulting in an anti-HCV prevalence of 39%. A total of 131 patients were HCV RNA-positive. HCV viremia was present in 63.5% of the anti-HCV-positive patients and in 10.3% of the anti-HCV-negative patients. Univariate analysis of risk factors showed that the number of previous blood transfusions, transfusion of blood before mandatory screening for anti-HCV, length of time on hemodialysis, and treatment in multiple units were associated with HCV positivity. However, multivariate analysis revealed that blood transfusion before screening for anti-HCV and length of time on hemodialysis were significantly associated with HCV infection in this population. These data suggest that nosocomial transmission may play a role in the spread of HCV in the dialysis units studied. In addition to anti-HCV screening, HCV RNA detection is necessary for the diagnosis of HCV infection in hemodialysis patients.

Real-time PCR-based quantification of Toxoplasma gondii in tissue samples of serologically positive outdoor chickens

This study aimed to quantify Toxoplasma gondii in tissue samples of serologically positive chickens using real-time polymerase chain reaction (PCR). Of 65 chickens evaluated, 28 were positive for T. gondii antibodies. Brain and heart samples were collected from 26 seropositive chickens and DNA was extracted using Trizol® and amplified using real-time PCR with SYBR® Green. Parasite DNA was detected in 24 of the 26 samples analyzed; the number of positive tissue samples and the parasite quantity did not differ between tissue types. The results confirmed the analytical sensitivity of parasite detection in chicken tissue samples and demonstrated the possibility of using other molecular systems for genotypic analysis.

Detection of human bocavirus and human metapneumovirus by real-time PCR from patients with respiratory symptoms in Southern Brazil

The introduction of newer molecular methods has led to the discovery of new respiratory viruses, such as human metapneumovirus (hMPV) and human bocavirus (hBoV), in respiratory tract specimens. We have studied the occurrence of hMPV and hBoV in the Porto Alegre (PA) metropolitan area, one of the southernmost cities of Brazil, evaluating children with suspected lower respiratory tract infection from May 2007-June 2008. A real-time polymerase chain reaction method was used for amplification and detection of hMPV and hBoV and to evaluate coinfections with respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, human rhinovirus and human adenovirus. Of the 455 nasopharyngeal aspirates tested, hMPV was detected in 14.5% of samples and hBoV in 13.2%. A unique causative viral agent was identified in 46.2% samples and the coinfection rate was 43.7%. For hBoV, 98.3% of all positive samples were from patients with mixed infections. Similarly, 84.8% of all hMPV-positive results were also observed in mixed infections. Both hBoV and hMPV usually appeared with RSV. In summary, this is the first confirmation that hMPV and hBoV circulate in PA; this provides evidence of frequent involvement of both viruses in children with clinical signs of acute viral respiratory tract infection, although they mainly appeared as coinfection agents.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.

A specific polymerase chain reaction method to identify Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a multidrug-resistant nosocomial pathogen that is difficult to identify unequivocally using current methods. Accordingly, because the presence of this microorganism in a patient may directly determine the antimicrobial treatment, conventional polymerase chain reaction (PCR) and real-time PCR assays targeting 23S rRNA were developed for the specific identification of S. maltophilia. The PCR protocol showed high specificity when tested against other species of Stenotrophomonas, non-fermentative Gram-negative bacilli and 100 clinical isolates of S. maltophilia previously identified using the Vitek system.

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture

Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

A novel indicator reaction for the catalytic determination of V(V) at ppb levels by the kinetic spectrophotometric method

A novel sensitive and relatively selective kinetic method is presented for the determination of V(V), based on its catalytic effect on the oxidation reaction of Ponceau Xylydine by potassium bromate in presence of 5-sulfosalicylic acid (SSA) as activator. The reaction was monitored spectrophotometrically by measuring the decrease in absorbance of Ponceau Xylydine at 640 nm between 0.5 to 7 min (the fixed time method) in H3PO4 medium at 25ºC. The effect of various parameters such as concentrations of H3PO4, SSA, bromate and Ponceau Xylydine, temperature and ionic strength on the rate of net reaction were studied. The method is free from most interferences, especially from large amounts of V(IV). The decrease in absorbance is proportional to the concentration of V(V) over the entire concentration range tested (1-15 ng mL−1) with a detection limit of 0.46 ng mL-1 (according to statistical 3Sblank/k criterion) and a coefficient of variation (CV) of 1.8% (for ten replicate measurement at 95% confidence level). The proposed method suffers few interferences such as Cr(VI) and Hg(II) ions. The method was successfully applied to the determination of V(V) in tap water, drinking water, bottled mineral water samples and a certified standard reference material such as SRM-1640 with satisfactory results. The vanadium contents of water samples were also determined by FAAS for a comparison. The recovery of spiked vanadium(V) was found to be quantitative and the reproducibility was satisfactory. It was observed that the results of the SRM 1640 were in good agreement with the certified value.

Detection and dynamics of porcine circovirus 2 shedding in semen using conventional and real-time PCR

The dynamics of porcine circovirus type 2 (PCV2) shedding in semen of naturally infected boars was studied. Semen was collected serially each 15 or 20 days during 62 days from 5 boars from a herd and from 11 boars from an artificial insemination center. All boars were positive for PCV2 DNA by nested polymerase chain reaction of raw semen in at least two sampling dates, and most of them had detectable shedding in all sampling dates. Real-time quantitative PCR was performed in 23 samples. All samples showed low amounts of PCV2 DNA, ranging from 98 to 652 PCV2 copies/mL. No differences between the frequencies of PCV2 DNA shed in semen were found considering herds and age of boars. PCV2 shedding in the semen can occur continuously or intermittently up to 60 days in naturally infected boars at 12 to 42 months old in absence of PCV2 clinical signs. These results demonstrate sporadic and long-term shedding patterns of low amounts of PCV2 DNA in semen from naturally infected boars.

Serology, polymerase chain reaction and histopathology for leptospirosis in samples collected at slaughter from dairy cows of Parnaiba region, state of Piauí, Brazil

The presence of anti leptospiral agglutinins (microscopic agglutination test - MAT) and DNA of leptospires was investigated in the kidney and urine (Polymerase Chain Reaction - PCR) in samples collected at the time of slaughter of cattle originating from the dairy basin of Parnaíba, Piauí, Brazil, as also the lesions in kidney, lung, liver, uterus, ovary and placenta (histopathology and immunohistochemistry). In the MAT, Hardjo was the predominant serovar with the highest number of reagent animals for the strain Hardjobovis/Sponselee. Anti-leptospiral antigens were scored in epithelial cells, interstitial vascular endothelium, endothelium of glomerular capillaries and Bowman's capsule of 20 positive animals. Inflammatory cells were more common in the kidney. PCR was positive in urine and kidney tissue

Development of a Real-time PCR test for porcine group A rotavirus diagnosis

Group A Rotavirus (RVA) is one of the most common causes of diarrhea in humans and several animal species. A SYBR-Green Real-Time polymerase chain reaction (PCR) was developed to diagnose RVA from porcine fecal samples, targeting amplification of a 137-bp fragment of nonstructural protein 5 (NSP5) gene using mRNA of bovine NADH-desidrogenase-5 as exogenous internal control. Sixty-five samples were tested (25 tested positive for conventional PCR and genetic sequencing). The overall agreement (kappa) was 0.843, indicating 'very good' concordance between tests, presenting 100% of relative sensitivity (25+ Real Time PCR/25+ Conventional PCR) and 87.5% of relative sensitivity (35- Real Time PCR/40- Conventional PCR). The results also demonstrated high intra- and inter-assay reproducibility (coefficient of variation ≤1.42%); thus, this method proved to be a fast and sensitive approach for the diagnosis of RVA in pigs.