Journal of Life Sciences Volume 6 Number (5)
J LS
Journal of Life Sciences
Volume 6, Number 5, May 2012 (Serial Number 49)
Contents
Biochemical and Microbiology
469 The Hemochromatosis Distribution in Matera Province: A New SNP to Explain the Low Genotype-Phenotype Correlation
Maria Carmela Padula, Marilena Larocca, Rocco Rossano, Luigi Milella, Domenico Dell’Edera and Giuseppe Martelli
A Survey on the Methods of Primer Design Among Plant Pathologists in Australia and New Zealand
Francisco M. Ochoa Corona, Brendan Rodoni and Joe Tang
481 Low Sero-Prevalence of Toxoplasma gondii IgG and IgM Antibodies in HIV Sero-Positive Patients
Attending National Hospital, Abuja, Nigeria
Tatfeng Youtchou Mirabeau and Adesua Ojo Ebikade
Development of Bioinfo-Portal Tool for the Analysis of Genomic and Proteomic Data
Rana Rehan Khalid, Bilal Hussain, Muhammad Ali, Muhammad Sajjad Ahmad, Asma Haque and Hira Qamar 489
Prevalence of Anti-HCV Antibodies Among Thalassemia Patients in Mosul City, Iraq
Mohammed D. Khalid and Basima A. Abdullah
492 Comparative Evaluation of Mannan Oligosaccharides and Acidifier Calcium Formate on the
Quail Digestive Tract
Eleftherios Bonos, Efterpi Christaki, Nikolaos Soultos, Amin Abrahim and Panagiota Florou-Paneri
Botany and Zoology
Ecology, Biology and Biometry of an Endemic Fabaceae: Genista Saharae Cosson and Durieu
Meriane Djamila and Kaabache Mohamed
Identification of the Far Eastern Species of Laminaria Lamouroux and Saccharina Stackhouse (Laminariales, Phaeophyceae) Based on Morphological Features
Olga N. Selivanova
The Effect of Weather and Agronomic Practice on Deoxynivalenol Mycotoxin in Durum Wheat
Isabel Giménez, Jacqueline Escobar, Elena Ferruz, Susana Lorán, Marta Herrera, Teresa Juan, Antonio Herrera and Agustín Ariño
518 Oribatid Use as Bioindicateur of Environment: Case of Galumna sp. and Scheloribates sp. (Acari: Oribatida)
Ghezali Djelloul and Harkat Hafsa
A Review of Recent Knowledge on Raptor Species in Sumatra, Indonesia
Hilda Zulkifli, Muhammad Iqbal, Adam A. Supriatna and Agus Nurza
The Best Dose for Sterilisation of Greenhouse Whitefly, Trialeurodes vaporariorum (Westwood) (Hem.: Aleyrodidae) by Gamma Radiation
Maryam Moradi and Mehdi Zarabi
Interdisciplinary Researches
543 Wastewater Treatment in the Oasis of Figuig (Morocco) by Facultative Lagoon System:
Physico-Chemical and Biological Aspect
Ouafae El Hachemi, Hassan Elhalouani, Antonina Torrens Armengol and Miquel Salgot
The Comparison of Amaranth Decolorization Ability for Two Types of Biological Consortia
Yovana Todorova, Mihaela Kirilova, Raycho Dimkov and Yana Topalova
The Long Term Evolution of Phosphates from the Cambic Chernozem at ARDS Caracal, Romania
Ana Maria Dodocioiu, Romulus Mocanu and Marian Dobre
The Research on Carbon Accumulation of Grassland Ecological System in China
Tao Li, Lei Ji, Jianrong Tan, Tao Liu, Zhongqi Song, Shujing Yang and Youmin Gan
Study of Contributing Factors for Cure Response in Patients with Acute Myeloblastic Leukemia (AML)
Saharnaz Ahmadi, Mostafa Rezaei-Tavirani, Adeleh Divsalar, Soheila Khodakarim and Leila Tahmasebi
Listeria monocytogenes in Live Mytillis galloprovincialis Collected from Butrinti Lagoon Located in South Part of Albania
Kapllan Sulaj, Halit Memoci, Xhuljeta Hamiti, Kastriot Korro and Fejzo Selami
Depression in End Stage Renal Disease: Comparison Between Patients Treated with Hemodialysis and Peritoneal Dialysis
Dragan Klaric and Vera Klaric
Journal of Life Sciences 6 (2012) 469-475
The Hemochromatosis Distribution in Matera Province:
A New SNP to Explain the Low Genotype-Phenotype Correlation
Maria Carmela Padula, Marilena Larocca, Rocco Rossano, Luigi Milella, Domenico Dell’Edera and Giuseppe Martelli Department of Biology, University of Basilicata, Potenza, Viale dell’Ateneo Lucano 85100, Italy
Received: November 24, 2011/Accepted: January 13, 2012/Published: May 30, 2012.
Abstract: The present study aims to investigate the genotype-phenotype correlation of the hereditary hemochromatosis (HH), a genetic disorder of iron metabolism, in Matera province (Basilicata, Italy). Integrating both epidemiological and molecular approaches, the authors studied: (a) the frequency of the HH main mutations; (b) the association between mutations and HH cases. The majority of patients with HH are homozygous for the C282Y mutation of the HFE gene. A second mutation (H63D) is more widely distributed and its connection with HH isn’t clear, but a low penetrance is attributed to this variant. The population-based study consists of three steps: (1) determination of iron biochemical parameters, (2) genetic test, and (3) sequencing of HFE gene and bioinformatics studies. A case report is presented in a 41-year-old male (genotype: H63D/wt) with biochemical and clinical evidences of HH, in absence of secondary iron overload factors. In the cohort of studied patients (150M:62F), there are 18 homozygous patients; H63D/H63D genotype is found in 11 cases. In the heterozygous group, H63D/wt is the predominant genotype (61/68 subjects). All the H63D/wt residents in the same village (Mont.) show altered biochemical parameter levels. In our case study,
a substitution localized into the HFE promoter (nt225A > C) is found. Results show that the H63D genotype is responsible for most cases of HH. The peculiar clinical manifestation found in Mont. suggests a founder effect. In our case, the iron overload is related to a presence of an undetected mutation, critical for the transcriptional regulation of the HFE gene.
Key words: Hereditary hemochromatosis, HFE gene variants, clinical phenotype, transcriptional regulation.
1. Introduction persistent fatigue and arthralgia, can appear; after a phase of latency, the first signs of biochemical
Hemochromatosis type 1 is an autosomal recessive expression appear, with a serum iron parameters disorder that occurs predominantly in Northern increase (serum transferrin, iron and ferritin) [5]. The European populations, with a prevalence of clinical manifestation occurs around the age of 40 in approximately 3-8 in 1,000 [1, 2]. HH is characterized males and later in females, because of the protective by excessive iron absorption, which progressively effects of menstrual blood loss and pregnancies [6]. leads to multi-organ failure. In fact, if untreated, The clinical expression occurs more frequently in hemochromatosis patients develop hepatic cirrhosis, males than in females (sex ratio of 3:1). HH can be hepatocellular carcinoma, cardiomyopathy, treated by phlebotomies or chelation therapy [7]. arrhythmias, diabetes, arthritis and hypogonadism [3, The identification of the HFE gene [8] at 6p21.3 4]. At an early stage, non-specific symptoms, enabled pre-symptomatic or earlier diagnosis [9].
Initially, the diagnosis relied on the liver biopsy Corresponding author: Maria Carmela Padula, Ph.D.
associated with evidence of elevated iron values [10]. candidate, research fields: molecular biology, human genetic.
E-mail: [email protected]. The genetic test is now widely available; about twenty
The Hemochromatosis Distribution in Matera Province: A New SNP to
Explain the Low Genotype-Phenotype Correlation
different mutations have been identified in the HFE altered iron metabolism or individuals belonging to gene worldwide, but the main mutations are known as
families with clinical evidence of iron overload. C282Y (exon4, nt845G > A; Cys282Tyr) and H63D
Biochemical parameters related to iron metabolism (exon2, nt187C > G; His63Asp) [11]. The HFE gene
(serum iron, serum transferrin and serum ferritin) are encodes the HFE protein, a transmembrane determined by standard biochemical methods glycoprotein implicated in the iron uptake modulation.
(including collection of serum after a 12-hour fast, It forms a heterodimeric complex with confirmation by at least two measurements). Serum β2-microglobulin [12, 13]. The C282Y disrupts a
transferrin saturation value is calculated as follows: disulfide bond in the α3 domain leading to lack of
[serum iron/(serum transferrin × 1.2)] × 100. HFE- β2microglobulin association [14].
The subsequent analysis of mutations related to The H63D mutation is responsible for the lack of
hemochromatosis relies on amplification of the HFE protein-transferrin receptor complex [15], a
specific gene region by PCR, followed by mutation critical interaction for the iron homeostasis detection using reverse dot-blot (Nuclear Laser maintenance.
Medicine S.r.l. kit). In particular, the methodology Although the HFE protein role in the iron
includes three steps: (1) DNA isolation, (2) PCR regulation is still unclear, several recent studies have
amplification using biotinylated primers, (3) emphasized a role in the regulation of intestinal iron
hybridation of amplification products to a test strip absorption by forming complexes with: (a) transferrin
containing allele-specific oligonucleotide probes receptor 1 (TfR1), in case of iron deficiency, or (b)
immobilized as an array of parallel lines. Bound transferrin receptor 2 (TfR2), the TfR1 liver homolog,
biotinylated sequences are detected using in case of iron overload. HFE/TfR2 complex actives a
streptavidin-alkaline phosphatase and color substrates. signaling cascade resulting in the upregulation of
The assay covers 11 HFE gene mutations (V53M, hepcidin and, consequently, a decreased dietary iron
V59M, H63D, H63H, S65C, Q127H, E168X, E168Q, uptake [16, 17].
W169X, C282Y, Q283P), four transferrin receptor Regarding the distribution of genotypes among iron
mutations (Y250X, E60X, M172K, the AVA Q loaded patients, most subjects are homozygous for
594-597) and 2 Ferroportin mutations (N144H, V162). C282Y mutation [18]. The percentages are > 90% in
To evaluate the quality and quantity of extracted the UK and Brittany, > 80% in Northern European
DNA two methods are used: (1) gel electrophoresis countries; it ranges from 60% and 83% in the USA
(1% agarose gel) in presence of standard at known [19, 20]. The C282Y/C282Y percentage in Italy is
concentration, and (2) spectrophotometric determination 64%; C282Y homozygous shows more severe iron
by means of NanoDrop™ spectrophotometer. overload than the other HFE genotypes [21]. The
In vitro amplification PCR is carried out, preparing H63D role in iron overload is controversial, but this
for each sample, a PCR mix: 15 µL amplification mix, condition rarely develops iron overload [22, 23].
5 µL Taq DNA polymerase, 5 µL DNA template. The Compound heterozygous C282Y/H63D has a thermocycling program is the following: (a) pre-PCR: mild-moderate phenotype [6].
94 °C/2 min; (b) thermocycling: 94 °C/15 sec., -58 °C/30 sec., -72 °C/30 sec. (35 cycles); (c) final
2. Material and Methods
extension: 72 °C/3 min. Amplification products are The present investigation is conducted in Basilicata,
analyzed by 3% agarose gel electrophoresis (fragment
a region of about six hundred thousand inhabitants lengths: 118, 169, 183, 201, 247, 287, 310, 347 bp). located in the South of Italy. The study includes
As for hybridization step on nitrocellulose strips, for patients who have or had some symptoms related to an
each polymorphic strip position, one of the staining
The Hemochromatosis Distribution in Matera Province: A New SNP to
Explain the Low Genotype-Phenotype Correlation
patterns shown in Table 1 should be obtained. isolated from the gel using sterile needles directly If the DNA test confirms the presence of mutations
placed in the band, after exposure to UV light. The in a patient, family testing is proposed. It combines
DNA isolated fragments are amplified in a final the collection of clinical, biochemical and genotypic
amplification mix volume equal to 50 µL, to verify the information for the patient relatives.
quality and uniqueness. The final step consists in HFE In order to better investigate the correlation
fragment sequencing. The next bioinformatics analysis between genetic background and clinical phenotype,
allows to verify the similarity between our sequences
a second level genetic analysis is performed, and HFE sequences reported in database. In particular, considering a case-model: 41-year-old male with
a multiple alignment is performed to identify some early clinical signs of iron overload, such as
conserved/non conserved nucleotides and fatigue, weakness, joint and abdominal pains and
consequently possible new variants. jaundiced complexion. To define the clinical picture, the patient is assayed for CBC, total bilirubin,
3. Results
transaminases, HBV and HCV serum markers, as Regarding the epidemiologic data, this study well as the iron biochemical parameters. Medical
involves 212 patients (median age: 53, range: 3-88), history is assessed to exclude additional risk factors
of whom 126 subjects (59%) are healthy, 68 (32%) are related to iron overload.
heterozygous (61 cases H63D/wt, 5 C282Y/wt, 2 The presence of unknown mutations in HFE gene
S65C/wt). There are 13 patients with homozygosity or an alteration of regulation mechanisms are
condition (11 cases H63D/H63D, 2 C282Y/C282Y). supposed to explain a low correlation. On the basis of
Five individuals are compound heterozygous (3 HFE sequences in NCBI database, the PCR primers
C282Y/H63D, 1 C282Y/S65C, 1 H63D/S65C). The are designed by means of NCBI Primer-Blast. After
sex ratio is equal to 2.42M:1F (150:62). In detail, the the optimization of reaction components and
authors observe the distribution shown in Table 2. The conditions, a series of amplifications is carried out to
Fig. 1 shows the distribution of the most important isolate and sequence gene fragments belonging to our
mutations related to the disease (C282Y and H63D) case DNA. For the amplification, 25 µL of PCR
within the population investigated. As H63D variant is
reaction are used: 1.5 µL MgCl 2 , each of dNTP 2 mM,
more frequent than C28Y in the province of Matera,
1 µL of specific primers, 0.4 U/µL of AmpliTaq we focus our attention on the distribution of this Gold ® DNA polymerase in 1× PCR buffer (100 mM variant: the Table 3 concerns the age distribution of
tris-HCl, pH 8.3, 500 mM KCl).The conditions of H63D genotypes and controls.
reaction are the following: (a) initial denaturation:
Table 2 Distribution of HFE genotypes within the analyzed
95 °C/7 min; (b) thermocycling: 94 °C/1 min; 58 °C/1
population.
min; 72 °C/2 min (43 cycles); (c) final extension:
Gender
HFE genotype
72 °C/10 min. Amplification products are analyzed by Male Female gel electrophoresis (1.5% agarose gel) in order to
H63D/wt 41
detect the most significant fragments. These ones are 0 S65C/wt 0 2
C282Y/wt 5
Table 1 Pattern for Genotype Identification.
H63D/H63D 4
C282Y/C282Y 2
Wild type line Mutant line Genotype
C282Y/H63D
1 0 Heterogyzous Positive
Normal Positive Negative Normal
C282Y/S65C
1 0 Homozygous Negative
Positive Heterozygous
H63D/S65C
Positive Homozygous
Wild-type 96
The Hemochromatosis Distribution in Matera Province: A New SNP to
Explain the Low Genotype-Phenotype Correlation
Fig. 1 Distribution of H63D and C282Y mutations in the analyzed population.
Table 3 Age distribution of H63D genotypes and controls.
HFE promoter region (GenBank ID: Y09801). Age (years) H63D/H63D H63D/wt
Controls
Up to 24 1 (0M:1F)
6 (3M:3F)
3 (2M:1F)
4. Discussion
25-34
0 3 (0M:3F) 11 (8M:3F)
Since the discovery of the HFE gene, several studies 45-54
35-44 4 (3M:1F)
8 (6M:2F)
21 (15M:6F)
have been performed in order to evaluate the H63D and 55-64
5 (2M:3F)
13 (11M:2F) 33 (30M:3F)
C282Y frequencies in different populations and to 65-74
1 (0M:1F)
11 (4M:7F)
16 (14M:2F)
provide information about the worldwide HFE 75 and older 0
1 (1M:0F)
13 (10M:3F) 30 (19M:11F)
6 (5M:1F)
12 (8M:4F)
mutations distribution [20, 24], but also about the genotype-phenotype correlation [25]. We have notified
Total 12
During data collection and interpretation, we have that no information is found about the H63D and identified a group of patients with a peculiar clinical
C282Y frequencies in Basilicata. In the present study behavior: some H63D carriers show abnormal iron
we perform a population-based study of the clinical biochemical parameter levels. They are inhabitants of
expression of the hemochromatosis gene, in order to: (a) the same village in Basilicata (Mont.). In order to
determine the main HFE mutation frequencies in investigate this particular behavior, we deeply study a
Matera district, (b) evaluate the correlation between peculiar case. We report the average (three genotype and phenotype in our population, and (c)
measurements) biochemical iron parameter values compare these results to other studies. observed in our clinical case: transferrin 312 mg/dL
About the first purpose, the epidemiological data (normal range: 200-360 mg/dL); ferritin: 948 ng/mL
show that the C282Y/C282Y genotype is found in two (normal range: 10-291 ng/mL); iron: 129 µL/dL
cases; we have no information on biochemical (normal range: 49-151 µL/dL).
parameters for these subjects. On the contrary, we About the results of the second level analysis,
observe a high frequency of H63D genotype; in some sequencing outcomes show a nucleotide change that
cases, the variation results in clinical could explain the clinical behavior anomaly found in
hemochromatosis. This data is not congruent with our case. In particular, applying a bioinformatics
previous studies in which are reported that H63D approach that integrates the similarity searches mutation does not result in iron overload in the (performed by BlastN of NCBI database) and the
absence of the C282Y mutation or other risk factors multialignment analysis (carried out using ClustalW2
[26, 27]. In particular, a positive correlation between Tool of EMBL-EBI database), we have established
H63D/H63D genotype and pathological iron that the change A > C resides in 225 position of the
parameters was found in 90% of cases. So these
The Hemochromatosis Distribution in Matera Province: A New SNP to Explain the Low Genotype-Phenotype Correlation
patients heal the iron overload with periodic blood donations. One of these subjects underwent liver transplantation, evidence of H63D/H63D genotype severity, in absence of other causes of hepatic injury. The majority of H63D/H63D patients are 35-55 years-old males, according to the late HH onset and to the sex differences.
Analyzing the compound heterozygous group (sex ratio of 4M:1F), we observe that the subject C282Y/H63D, a 54 years old male, treats the iron overload with periodic phlebotomy, suggesting a synergistic effect of the two mutations on the clinical HH expression. The C282Y/S65C individual shows a value of transferrin saturation equal to 32% (n.r. 15-45%); the TS value of H63D/S65C patient is equal to 24%. In this case we don’t emphasize the H63D genotype severity, because there are no significant differences between the two TS values: they are within the normal range both in absence (C282Y/S65C genotype) and in presence (H63D/S65C genotype) of H63D variant. However, we could relate this aspect to the patient age: they are under 30 years-old. Instead the clinical manifestation of HH occurs 10 years later and the subsequent clinical signs are due to the progressive alteration of the physiology of involved organs, liver in particular.
Within the H63D/wt genotype group, we observe a particular clinical manifestation that has revealed a
low genotype-phenotype correlation: about half of the subjects (28/61) show abnormal values of iron parameters in absence of other factors. In detail, focusing our attention on the distribution of these subjects in the analyzed population, we observed that, of these 28, 18 subjects are inhabitants of the same small village in Basilicata (Mont.) and all these individuals have biochemical evidences of iron overload (serum ferritin value higher than 750 ng/mL) without evidence of secondary iron overload causes. For this reason, we report and analyze the case-model of a male who lives in Mont. village, with H63D/wt genotype. To elucidate this experimental evidence, we sequence the HFE coding region, the 5’UTR (UnTranslated Region) and the 3’UTR. No mutation has been found in HFE CDS, 3’UTR; a new polymorphic site is individuated within the promoter region (nt225 A > C). A previous study provided information about the transcriptional regulation of the human HFE gene and defined the functional organization of the HFE promoter. The putative gene regulatory element site of the 5’-flanking region has been identified and we can localize our polymorphism two nucleotides downstream of the TATA binding protein (TBP) site [28], by aligning our case model and the RefSeq as shown in Fig. 2. This transcriptional factor is essential for the promoter activation and for the assembly of transcriptional complex [29].
Fig. 2 ClustalW2 output: alignment between HFE promoter RefSeq (GenBank ID: Y09801) and isolated HFE promoter sequence (EMO6_1).
The Hemochromatosis Distribution in Matera Province: A New SNP to
Explain the Low Genotype-Phenotype Correlation
Our polymorphic site resides in a regulatory region new mutation (nt225 A > C), never described before within the core promoter, important for the regulation
in literature, explains, at molecular level, the peculiar of the HFE gene. This aspect is very important because
HH clinical expression: we demonstrate that in our it is known that the regulation of gene expression is
clinical case, the iron overload is related to a promoter achieved through the interaction of several control
mutation that affects the transcriptional regulation of levels including the regulation of transcription the HFE gene.
initiation. In some cases, an association between promoter
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A Survey on the Methods of Primer Design Among Plant Pathologists in Australia and New Zealand
1 2 Francisco M. Ochoa Corona 3 , Brendan Rodoni and Joe Tang
1. Oklahoma State University, National Institute for Microbial Forensics & Food and Agricultural Biosecurity (NIMFFAB), 127 Noble Research Center, Stillwater, OK 74078-3003, USA 2. Department of Primary Industries, Knoxfield Centre, Private Bag 15, Ferntree Gully Delivery Centre, Victoria, Australia
3. Plant Health and Environment Laboratory, Investigation and Diagnostic Centre, MAF Biosecurity New Zealand, P.O. Box 2095,
Auckland 1140, New Zealand
Received: October 14, 2011/Accepted: December 12, 2011/Published: May 30, 2012.
Abstract: Designing primers for PCR-based diagnostics was achieved by executing sight searches on DNA sequences. Visual searching for specific DNA targets is time consuming, subjective and requires optimisation among numerous candidate primer sets. Several primer design software have been linked to useful bioinformatic packages to speed the development of PCR assays. Despite the software options available, primer design has remained a challenging aspect of incursion responses, biosecurity emergencies and microbial forensic applications. Two surveys were conducted among 45 plant virologists and 21 other plant pathologists during the 7th Australasian Plant Virology Workshop and the 16th Biennial Australasian Plant Pathology Conference in 2006 and 2007, respectively. Results show that most primer design learning occurs scientist to scientist rather than during academic teaching. This tendency matches with 16% of scientists users of PCR, who do not engage in primer design and 25% designing primers only by visual means, combining a pool of 41% who if trained, would likely enhance their performance in primer design. Only 13 out of 58 scientists ranked themselves as experts. Implementing primer design in study programs and regional training will benefit plant pathology and entomology, and the responsiveness and performance of biosecurity and microbial forensics in the South Pacific.
Key words: Primer design, PCR, education, training, biosecurity, microbial forensics, plant pathology.
1. Introduction assay was achieved. More recently, several primer design software packages have been developed, and
Since the first report of the polymerase chain serve as useful bioinformatic tools to speed the reaction (PCR) [1-4], oligonucleotide primer design development of PCR-assays [1, 5-9]. However, has become a cornerstone step for developing despite the number of options now available for PCR-based assays. The selection of oligonucleotide primer design, the rapid development of primer sequences with appropriate priming and sequences required for PCR detection of exotic thermodynamic characteristics was achieved initially micro-organisms during incursion responses, by executing visual searches of targeted DNA molecular detection, and other biosecurity and sequences followed by tedious and time consuming microbial forensic applications remain challenging for rounds of PCR optimization applied to numerous
a number of scientists.
primer set candidates until a robust and repeatable
A survey was conducted among plant pathologists in Australia and New Zealand, with the aim of Corresponding author: Francisco M. Ochoa Corona, Ph.D.,
obtaining insights on current capabilities and gaps, assistant professor, research fields: plant virology, microbial
forensics, diagnostics and detection, biosecurity. E-mail: and future training and educational needs to enhance [email protected].
A Survey on the Methods of Primer Design Among Plant Pathologists in Australia and New Zealand
biosecurity responsiveness in this region. This survey also explored how knowledge of primer design and new related developments in this field is communicated among scientists working on the development of detection methods, disease diagnostics, biosecurity, microbial forensics and general molecular biology.
2. Materials and Methods
Two surveys were conducted during the 2006 7th Australasian Plant Virology Workshop held in Rottnest Island, Australia, and the 2007 16th Biennial Australasian Plant Pathology Conference in Adelaide, Australia. The surveyed universe consisted of 66 interviews, 45 of which were of plant virologists and
21 of other plant pathologists. Eight interviews were discarded because of conflicts in the provided answers, leaving 58 interviews. The interviewed scientists were affiliated with universities (47%), government (37%), private industry (6%) and part time combinations such as universities-government (8.8%) and government-private (2.2%). The interviewed universe was composed of scientists (24.4%), graduate students (24.4%), academic faculty (15.5%), postdoctoral associates (6.6%), laboratory technicians (2.2%), managers (2.2%), as well as participants who did not state their occupation (24.4%). Moreover, 51.1% of respondents were virologists, 2.2% mycologists, 2.2% nematologists, and 44.5% did not specify an area of expertise. Other questions explored the preferred resources and methods used during primer design, such as how primer design skills were learned, and the willingness to share expertise in primer design with colleagues. A call to structure a list of experts in primer design was also included. All questions were provided in multiple choices format and percentages calculated after data collection. Percentages were rounded to the proximal decimal.
3. Results
The survey indicates that 25% of the surveyed
scientists use only visual analysis of targeted DNA sequences for primer design, while 11% use only software. Fifty eight percent combined both methods and 5% did not answer (Fig. 1). When these plant pathologists were asked about what they do when requiring primers, 48% indicated to be self-designers that eventually also picks primers from literature, 28% expressed to be only self-designers, and 8% design primers or rely on team members or contract services to get them. The rest 16% do not engage in primer design. In this group only 6% rely on team members expertise, 4% may rely on team members but also select primers from literature, 2% only select primers from literature, 2% only contract services, and 2% do not know how (Fig. 2). Regarding how primer design skills are gained, 7% learned during undergraduate or graduate education, 14% during postdoctoral research, 28% through colleagues, 22% were self learners and 29% learned by themselves and with colleagues combining more than one way of learning, but none had learned during workshops or conferences. All of whom group 79% of self learners scientists that learned with colleagues, or by combining different learning approaches, for example combining either with colleagues and other approaches or by self- learning plus another approach, on the contrary,
Fig. 1 Preference for primer design methods.
Twenty five percent of the surveyed scientist use only visual analysis of DNA sequences for primer design, 11% uses only software. Fifty eight percent combine both methods and 5% did not answer.
Only visual analysis 25%
visual & software 58%
No
answered 5%
A Survey on the Methods of Primer Design Among Plant
Pathologists in Australia and New Zealand
Literature or
Self ‐ design or
relay on team
relay on team
member or contract
them
8% Self ‐ design 28%
Self ‐ design &
Only literature
literature 2%
Only Only relay in Don’t contract team
know how
Fig. 2 How scientists as users of PCR obtain primer sequences.
Sixteen percent do not engage in primer design (6% relay on team members expertise, 4% may rely on team members but also select primers from literature, 2% only select primers from literature, 2% only contract services, and 2% do not know how) in contrast to 48% that either design or pick primers from literature. Twenty eight percent claimed to only design the primers they need.
1.78 1.78 5.3% Post ‐ graduate
With colleagues
course
& self ‐learner
1.78 Doctoral Res.
1.78 1.78 Workshops/Conf.
with colleagues
Self ‐learner
Fig. 3 How scientist learned primer design.
Seventy nine percent of surveyed scientists comprise self learners, scientists that learned with colleagues, or scientist combining different approaches for learning (combining either with colleagues and other approach or by self-learning plus another approach), in contrast to 21% who had learned during postdoctoral research, undergraduate or graduate studies. No learning during attendance at scientific conferences or meetings was reported.
21% had learned during formal education less competitive, 7% think that sharing expertise (postdoctoral research, undergraduate or graduate
would make them less competitive and 19% did not studies) (Fig. 3). When asked about their willingness
answer. Only 13 out of the 58 scientists interviewed at to share expertise and whether sharing their the two conferences ranked themselves as experts. expertise.or skills could make them less competitive,
Seven of these were from Australia, three from New 72% were willing to share their expertise, 14% were
Zealand and the remaining three respondents were not, and 14% did not answer. Similarly, 72% did not
from South Africa, the UK and the USA. The software think that sharing their expertise would make them
used by scientists to assist the design of primer
A Survey on the Methods of Primer Design Among Plant
Pathologists in Australia and New Zealand
sequences include (in alphabetical order): Amplify 1.2, Similarly, 14% of these plant pathologists rely on Gprime, DNAMAN, Oligo, Primer Express, Primer3,
team members or contract services for primer design Primo, Premier3, PCR/DNA detective [1, 5-8, 10, 11].
or select primers from the literature (Fig. 2). The results obtained from PCR based procedures
4. Discussion
during initial phases of detection and investigation are
A number of interpretations can be derived from of fundamental value to downstream the presumptive this survey. However, the aim of this survey was to
diagnosis that lead to a definitive diagnosis [12]. obtain elements for discussion toward training and
Design of optimal primers is paramount for the education needs, and the implementation of measures
successful development of effective PCR detection for improving primer design as a catalytic factor for
procedures. This survey shows that improvements in the improvement of responsiveness and awareness of
primer design performance can be gained by biosecurity and microbial forensics in the South
incorporating primer design principles in plant Pacific. The surveyed plant pathologists were not
pathology, entomology, microbial forensics and equally comfortable with sharing some information.
biosecurity training programs.
For example, 44.5% did not provide details about their Primer design strategies comprise a relatively new specialty field and 24.4% of the respondents declined
area of knowledge and skills, and involve a to provide details about their type of appointment.
conglomerate of knowledge from molecular biology, Some survey questions not answered by the whole
bioinformatics, thermodynamics, biochemistry, universe of interviewed scientists were not evaluated
mathematics and chemistry among other basic during this survey (not shown). Yet, the information
sciences. Techniques of primer design can be fit obtained depicts the preferences of a group of
logically into a variety of curricula as either a scientists in Australia and New Zealand regarding
curriculum component or part of course works in plant methods used for primer design, and for learning and
pathology, entomology, forensics and biosecurity. communicating their expertise. Two major tendencies
5. Conclusions
were observed regarding how primer design skills are learned. Seventy nine percent of surveyed scientists
This survey showed that 74% of a selected group of describe themselves as self learners, or learned with
plant pathologists in the South Pacific are willing to colleagues, or combined these two learning share their primer design expertise, and at least ten approaches (Fig. 3), on the contrary, 21% had learned
self-described primer design experts are located in this during postdoctoral research, undergraduate or region. Incorporation of primer design into curricula graduate studies (Fig. 3). No learning during and study programs and the implementation of attendance at scientific conferences or meetings was
regional training workshops will benefit current reported (Fig. 3). This result suggests that most of the
programs in plant pathology and entomology, and will primer design learning occurs scientist to scientist
improve the responsiveness and performance of rather than during academic teaching. These programs in biosecurity, microbial forensics in the tendencies match with additional results indicating
South Pacific.
that 16% of these scientists who are users of PCR, do
Acknowledgments
not engage in formal primer design (Fig. 2) and 25% design primers only by visual means (Fig. 1),
The authors acknowledge Professors Jacqueline combining a pool of 41% who if trained, would likely
Fletcher and Li Ma, Oklahoma State University, enhance their level of performance in primer design.
Department of Entomology and Plant Pathology, also
480
A Survey on the Methods of Primer Design Among Plant
Pathologists in Australia and New Zealand
affiliated to the National Institute for Microbial [6] A. Gibbs, J. Armstrong, A.M. Mackenzie, G.F. Weiller, The GPRIME package: Computer programs for
Forensics & Food and Agricultural Biosecurity identifying the best regions of aligned genes to target in
(NIMFFAB) for the review of the manuscript and nucleic acid hybridisation-based diagnostic tests, and valuable comments.
their use with plant viruses, Journal of Virological Methods 74 ( 1998 ) 67-76.
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Low Sero-Prevalence of Toxoplasma gondii IgG and IgM Antibodies in HIV Sero-Positive Patients Attending National Hospital, Abuja, Nigeria
1 Tatfeng Youtchou Mirabeau 2 and Adesua Ojo Ebikade
1, Department of Medical Laboratory Science, College of Health Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria 2, Department of Chemical Pathology, National Hospital, Abuja, Nigeria
Received: September 04, 2011/Accepted: October 22, 2011/Published: May 30, 2012.
Abstract: Several researchers have investigated the association of numerous opportunistic pathogens with HIV, little is documented on its association with T. gondii in our environment. We investigated the prevalence of T. gondii immunoglobulins G and M (IgG
and IgM) in HIV positive individuals in relation to their cluster of differentiation 4 (CD 4 ) cells count. IgG, IgM and CD 4 were assayed using enzyme immunoassay (EIA) and flowcytometry respectively. 341 HIV positive individuals were studied in the present research, 30 (8.7%) of them had T. gondii IgG and IgM, 297 subjects had CD 4 cells count a range of 200-400 cells/ μL, 27 (9.7%) and 2 (0.6%) of which had T. gondii IgG and IgM respectively. Of the 44 HIV positive subjects with CD 4 > 400 cells/ μL, one (2.2%) was positive for T. gondii IgG. In the control group, all the 177 had CD 4 > 400 cells/ μL of which, one (0.5%) had T. gondii IgG. The prevalence of T. gondii infection was significantly higher in HIV positive individuals than in controls (P < 0.05). Male subjects in the age bracket 18-30 years had significantly higher prevalence when compared to other groups (P < 0.05). Although the present findings revealed a low prevalence of T. gondii antibodies in HIV infection, this suggests that a differential toxoplasmosis diagnosis is also necessary in cases of encephalitis in HIV infection.
Key words: Toxoplasma gondii, IgG, IgM, CD 4 , HIV-sero-positive.
1. Introduction cell-mediated immunity is at risk for reactivation of the infection [1, 2]. Toxoplasmosis in this setting manifests
Toxoplasma gondii is an obligate intracellular primarily as toxoplasmic encephalitis. The prevalence protozoan of worldwide distribution. Development of of serologic evidence of T. gondii infection varies cell-mediated immunity after acute infection with T. depending on geographic locale and population group. gondii results in control but not eradication of the About 3%-67% of adults in the United Sates are infection [1]. The ensuing chronic or latent phase of sero-positive for antibodies against T. gondii [1]. The infection is characterized by the persistence of the rate of seroprevalence can be as high as 90% in organism in tissues of the infected individual Western Europe and tropical countries. (primarily brain, skeletal muscle, and heart). Indeed, T. Transmission to humans occurs primarily by gondii is one of the most common causes of chronic ingestion of undercooked pork or lamb meat that infection with an intracellular organism in humans. A contains tissue cysts or by exposure to oocysts either chronically infected individual who develops defects in through ingestion of contaminated vegetables or direct
contact with cat feces [1]. Other modes of transmission Corresponding author: Tatfeng Youtchou Mirabeau, Ph.D.,
research fields: malaria, HIV, immunology. E-mail: include the transplacental route, blood product [email protected].
Low Sero-Prevalence of Toxoplasma gondii IgG and IgM Antibodies in HIV Sero-Positive Patients Attending National Hospital, Abuja, Nigeria
transfusion, and organ transplantation. Acute infection
were excluded.
in immunocompetent individuals is usually Verbal consent of the patients was sought and asymptomatic.
ethical approval was obtained from the management Toxoplasmic encephalitis usually occurs in of the National Hospital Abuja.
HIV-infected patients with CD 4 T-cell counts < 100/µL
2.3 Sample Analysis
[2]. T. encephalitis in AIDS patients in the United States is almost always caused by reactivation of a
The blood collected in the plane containers was chronic infection. Thus, the incidence of this disease