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Identification of hybrids of painted and milky storks using FTA card-collected blood, molecular markers, and morphologies

Yee EY, Zainuddin ZZ, Ismail A, Yap CK, Tan SG

Biochem Genet, 2013 Oct;51(9-10):789-99.
PMID: 23846110 DOI: 10.1007/s10528-013-9607-8

Suspicious hybrids of painted storks and milky storks were found in a Malaysian zoo. Blood of these birds was sampled on FTA cards for DNA fingerprinting. Of 44 optimized primers, 6 produced diagnostic markers that could identify hybrids. The markers were based on simple, direct PCR-generated multilocus banding patterns that provided two sets of genetic data, one for each of the two stork species and another for the hybrids. It also revealed that large DNA fragments (3,000 bp) could be amplified from blood collected on FTA cards. When the results of each individual bird's DNA fingerprint were compared with plumage characters, the hybrids were found to express a range of intermediate phenotypic traits of the pure breeds with no dominant plumage characteristic from either parental species.

Matched MeSH terms: DNA Primers
The genetic structure of Oreochromis spp. (Tilapia) populations in Malaysia as revealed by microsatellite DNA analysis

Bhassu S, Yusoff K, Panandam JM, Embong WK, Oyyan S, Tan SG

Biochem Genet, 2004 Aug;42(7-8):217-29.
PMID: 15487586

The genetic make-up of five populations of Oreochromis spp. was examined by microsatellite analysis. Eleven polymorphic microsatellite loci showed significant departures from the Hardy-Weinberg equilibrium. The mean heterozygosity ranged from 0.6280 to 0.7040 for each population. The genetic distance values showed a clear separation between O. niloticus and O. mossambicus. The differentiation of the O. niloticus populations was then tested with various genetic measures, which are based on both the Infinite Allele and the Stepwise Mutation models. All these measures grouped the populations similarly.

Matched MeSH terms: DNA Primers
Identification and characterization of Malaysian river catfish, Mystus nemurus (C&V): RAPD and AFLP analysis

Chong LK, Tan SG, Yusoff K, Siraj SS

Biochem Genet, 2000 Apr;38(3-4):63-76.
PMID: 11100266

This work represents the first application of the amplified fragment length polymorphism (AFLP) technique and the random amplified polymorphic DNA (RAPD) technique in the study of genetic variation within and among five geographical populations of M. nemurus. Four AFLP primer combinations and nine RAPD primers detected a total of 158 and 42 polymorphic markers, respectively. The results of AFLP and RAPD analysis provide similar conclusions as far as the population clustering analysis is concerned. The Sarawak population, which is located on Borneo Island, clustered by itself and was thus isolated from the rest of the populations located in Peninsular Malaysia. Both marker systems revealed high genetic variability within the Universiti Putra Malaysia (UPM) and Sarawak populations. Three subgroups each from the Kedah, Perak, and Sarawak populations were detected by AFLP but not by RAPD. Unique AFLP fingerprints were also observed in some unusual genotypes sampled in Sarawak. This indicates that AFLP may be a more efficient marker system than RAPD for identifying genotypes within populations.

Matched MeSH terms: DNA Primers
Fulltext A pentaplex PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus and Panton-Valentine Leucocidin

Al-Talib H, Yean CY, Al-Khateeb A, Hassan H, Singh KK, Al-Jashamy K, et al.

BMC Microbiol, 2009;9:113.
PMID: 19476638 DOI: 10.1186/1471-2180-9-113

Staphylococcus aureus is a major human pathogen, especially methicillin-resistant S. aureus (MRSA), which causes a wide range of hospital and community-acquired infections worldwide. Conventional testing for detection of MRSA takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern.

Matched MeSH terms: DNA Primers
Fulltext Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens

Nik Zuraina NMN, Goni MD, Amalina KN, Hasan H, Mohamad S, Suraiya S

Diagnostics (Basel), 2021 Apr 22;11(5).
PMID: 33922299 DOI: 10.3390/diagnostics11050753

A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.

Matched MeSH terms: DNA Primers
Fulltext Surface display of glycosylated Tyrosinase related protein-2 (TRP-2) tumour antigen on Lactococcus lactis

Kalyanasundram J, Chia SL, Song AA, Raha AR, Young HA, Yusoff K

BMC Biotechnol, 2015;15:113.
PMID: 26715153 DOI: 10.1186/s12896-015-0231-z

The exploitation of the surface display system of food and commensal lactic acid bacteria (LAB) for bacterial, viral, or protozoan antigen delivery has received strong interest recently. The Generally Regarded as Safe (GRAS) status of the Lactococcus lactis coupled with a non-recombinant strategy of in-trans surface display, provide a safe platform for therapeutic drug and vaccine development. However, production of therapeutic proteins fused with cell-wall anchoring motifs is predominantly limited to prokaryotic expression systems. This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study, the glycosylated murine Tyrosinase related protein-2 (TRP-2) with the ability to anchor onto the L. lactis cell wall was produced in suspension adapted Chinese Hamster Ovary (CHO-S) cells by expressing TRP-2 fused with cell wall anchoring LysM motif (cA) at the C-terminus.

Matched MeSH terms: DNA Primers
Fulltext Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample

Foo PC, Nurul Najian AB, Muhamad NA, Ahamad M, Mohamed M, Yean Yean C, et al.

BMC Biotechnol, 2020 Jun 22;20(1):34.
PMID: 32571286 DOI: 10.1186/s12896-020-00629-8

BACKGROUND: This study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model.
RESULTS: A set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites.
CONCLUSIONS: The analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

Matched MeSH terms: DNA Primers
Fulltext Enhancers of Agrobacterium-mediated transformation of Tibouchina semidecandra selected on the basis of GFP expression

Thau, Wilson Lym Yon, Henry, Erle Stanley, Janna Ong Abdullah

Trop Life Sci Res, 2010;21(2):-.
MyJurnal

Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic
acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 µM galactose and 100
µM tyrosine supplemented with 600 µM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 µM galactose and 50 µM tyrosine with 200 µM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative
T. semidecandra transformants was verified by PCR amplification with specific primers.

Matched MeSH terms: DNA Primers
Fulltext The Effect of Oxidative Stress Towards The Expression of Thiamine Biosynthesis Genes (THIC and THI1/THI4) in Oil Palm (Elaeis guineensis)

Idris ZHC, Abidin AAZ, Subki A, Yusof ZNB

Trop Life Sci Res, 2018 Mar;29(1):71-85.
PMID: 29644016 MyJurnal DOI: 10.21315/tlsr2018.29.1.5

Thiamine is known to be an important compound in human diet and it is a cofactor required for vital metabolic processes such as acetyl-CoA biosynthesis, amino acid biosynthesis, Krebs and Calvin cycle. Besides that, thiamine has been shown to be involved in plant protection against stress. In this study, the level of expression of THIC and THI1/THI4, the genes for the first two enzymes in the thiamine biosynthesis pathway were observed when oil palm (Elaeis guineensis) was subjected to oxidative stress. Primers were designed based on the consensus sequence of thiamine biosynthesis genes obtained from Arabidopsis thaliana, Zea mays, Oryza sativa, and Alnus glutinosa. Oxidative stress were induced with various concentrations of paraquat and samplings were done at various time points post-stress induction. The expression of THIC and THI1/THI4 genes were observed via RT-PCR and qPCR analysis. The expression of THIC was increased 2-fold, while THI1/THI4 gene transcript was increased 4-fold upon induction of oxidative stress. These findings showed that oil palm responded to oxidative stress by over-expressing the genes involved in thiamine biosynthesis. These findings support the suggestion that thiamine may play an important role in plant protection against stress.

Matched MeSH terms: DNA Primers
DNA barcoding relates Trichuris species from a human and a man's best friend to non-human primate sources

Brandon-Mong GJ, Ketzis JK, Choy JS, Boonroumkaew P, Tooba M, Sawangjaroen N, et al.

Trop Biomed, 2018 Dec 01;35(4):1131-1139.
PMID: 33601860

Trichuris trichiura, the whipworm of humans, is one of the most prevalent soiltransmitted helminths (STH) reported worldwide. According to a recent study, out of 289 STH studies in Southeast Asia, only three studies used molecular methods. Hence, the genetic assemblages of Trichuris in Southeast Asia are poorly understood. In this study, we used partial mitochondrial DNA (cytochrome c oxidase subunit 1 or COI) sequences for analysis. Trichuris grouped in a same clade with different hosts indicate the potential of cross infection between hosts. Based on COI, the adult Trichuris isolated from a Malaysian patient was most closely related to Trichuris isolated from Papio anubis (olive baboons) from the USA. The Trichuris isolated from the dog from Malaysia was genetically similar to a Trichuris species isolated from Macaca silenus (lion-tailed macaque) from Czech Republic. Both the human and dog isolated Trichuris grouped in clades with different hosts indicating the potential of cross infection between hosts. Specific PCR primers based on the partial COI of T. trichiura isolated from African green monkey and T. serrata were designed and successfully amplified using multiplex PCR of the pooled DNA samples. Our results suggest a complex parasite-host relationship, and support the theory of cross infection of Trichuris between humans and non-human primates as suggested in previous publications.

Matched MeSH terms: DNA Primers
Fulltext Multiplex Polymerase Chain Reaction (PCR) efficiency in detection of pathogenic Escherichia coli O157:H7

Suria, M. S., Adlin Azlina, A. K., Mohd Afendy, A. T., Zamri, I.

International Food Research Journal, 2013;20(6):3307-3311.
MyJurnal

Shiga toxin-producing E. coli (STEC) is an important foodborne pathogen causing diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome in humans. STEC is an implicated in the vast majority of outbreaks, widely via consumption of STEC contaminated beef, as important vehicle of transmission of this organism to human. The E. coli O157:H7 serotype is traditionally identified by serological identification of the somatic antigen (O157) and structural flagella (H7). In this study, the bacteria were identified as STEC serotype O157:H7 with three primer pairs that amplified fragments of secD, rfbE and fliC genes in PCR assays. These primer pairs specifically amplified different sizes of target genes: a 244bp region of the E. coli diagnostic marker gene (secD); a 317bp region of the O157 lipopolysacharide (LPS) gene (rfbE); and a 381bp region of the H7 flagellin gene (fliC). The singleplex, duplex and triplex PCR assay developed in this study have a sensitivity limit at 2.8 x 103, 2.8 x 105 and 2.8 x 107 CFU/ml of E. coli O157:H7, respectively. Sensitivity to detect trace amount of E. coli O157:H7 DNA was reduced as the number of primer used was increased for competing to the same DNA template.

Matched MeSH terms: DNA Primers
Identification of the species origin of commercially available processed food products by mitochondrial DNA analysis

Chandrika, M., Maimunah, M., Zainon, M.N., Son, R.

International Food Research Journal, 2010;17(4):867-876.
MyJurnal

Legislation concerning the safety assessment and labelling of foodstuffs has been implemented in many countries. Consequential to a number of cases of food adulteration reported globally, a fast and reliable detection method for the food traceability is required in ensuring effective implementation of food legislation in a country. In this study, PCR-RFLP technique based on cyt b gene has been tested for its suitability for these purposes. This method combines the use of a pair of universal primer that amplifies a 359 bp fragment on the cyt b gene from meat muscle DNA and restriction enzyme analysis. Analysis of experimental beef frankfurter, minced beef, pork frankfurter and pork cocktail samples demonstrated the suitability of the assay for the detection of the beef (Bos taurus) and pork (Sus scrofa), but not applicable for some processed food, particularly detection of mackerel (Rasterelliger brachysoma), sardine (Saedinella Fimbriata) and tuna (Thunnus tonggol) origin in canned food. Commercial frauds through species mislabelling or misdescribed were not detected. The assay is demonstrated applicable for routine analysis of meat traceability of foodstuffs and legislation purposes, if sufficient availability of detectable mtDNA in the foodstuffs is ensured.

Matched MeSH terms: DNA Primers
DNA profiling among egg and beef meat isolates of Escherichia coli by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplified polymorphic DNA-PCR (RAPD-PCR)

Sahilah, A.M., Audrey, L.Y.Y., Ong, S.L., Wan Sakeenah, W.N., Safiyyah, S., Norrakiah, A.S., et al.

International Food Research Journal, 2010;17(4):853-866.
MyJurnal

Forty three (n=43) genomic DNA of Escherichia coli (11 isolates from eggs and 32 isolates from imported beef meats) were characterized by shiga toxin 1 (stx1), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplified polymorphic DNA-PCR (RAPD-PCR) analyses. In the shiga toxin 1 (stx1) gene detection with primer stx 1F (5’-TTCTTCGGTATCCTATTCCC-3’) and stx 1R (5’- CTGTCACAGTAACAACCGT-3’), 9 E. coli of beef meats isolates were positive toward sxt1 gene. The results of the ERIC-PCR and RAPD-PCR were analyzed using GelCompar II software. ERIC-PCR with primer ERIC1 (5’-CACTTAGGGGTCCTCGAATGTA -3’) and ERIC2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’) discriminated the E. coli into 6 clusters and 10 single isolates at 80% similarity. RAPD-PCR with primer Gen8 and Gen9, produced 10 clusters and 15 single isolates and 12 clusters and 14 single isolates of 80%, respectively. These results demonstrated that both ERIC-PCR and RAPD-PCR are useful and suitable tools for molecular typing of those isolates examined.

Matched MeSH terms: DNA Primers
Fulltext Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs

Kakuda T, Shojo H, Tanaka M, Nambiar P, Minaguchi K, Umetsu K, et al.

PLoS One, 2016;11(6):e0158463.
PMID: 27355212 DOI: 10.1371/journal.pone.0158463

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

Matched MeSH terms: DNA Primers
Fulltext Characterization of Edwardsiella tarda isolated from Asian Seabass, Lates calcarifer

Nadirah, M., Najiah M., Teng, S. Y.

International Food Research Journal, 2012;19(3):1247-1252.
MyJurnal

This study described the antibiotic and heavy metal resistance pattern of 17 isolates of Edwardsiella tarda obtained from Asian seabass (Lates calcarifer). E.tarda isolates were resistant to oleandomycin, lincomycin, novobiocin and spiramycin. In contrast, most of the isolates showed high level of susceptibility to tetracycline, doxycycline, florfenicol, chloramplenicol, nitrofurantoin, fosfomycin, kanamycin, oxolinic acid and flumequine. MAR value was 0.35 which indicated that the cultured Asian seabass have received high exposure to those tested antibiotics. Besides, very high level of heavy metal resistance among these isolates was observed. Genotypic profile of DNA fingerprintings generated by RAPD-PCR using M13 universal primer and M13 wild type phage primer showed high degree of genetic diversity with percentages similarity and genetic distance among the isolates were ranging from 10.5% to 100% and 0 to 0.895, respectively. This result indicates that strains that belong to the same origin were not always closely related genetically.

Matched MeSH terms: DNA Primers
Fulltext A simple protocol to isolate DNA from Malaysian stork blood collected on FTA® cards

YEE, ELSIE Y. S., ZAINAL ZAHARI, AHMAD ISMAIL, YAP, C.K., TAN, S. G.

Buletin Persatuan Genetik Malaysia, 2009;14(1):20-22.
MyJurnal

The blood of the Painted Storks (Mycteria leucocephala) and the Milky Storks (M. cinerea) from Malaysia were collected
invasively from the breeding site. The blood was dropped on to FTA® cards and stored at room temperature. DNA was isolated from
the FTA® cards through a modification of the Wizard DNA Purification kit (Promega) procedure and PCR was performed with 11 pairs
of microsatellite primers of the American Wood Stork (M. americana). The collection of a drop of blood onto the card is superior to the
usual practice of collecting about five ml of blood into a vacuum tube as it causes fewer traumas to these sensitive birds. Moreover, this
collection procedure can be adopted for use in various wild animal species which are usually found in the remote areas of Malaysia as
the sample collection cards can be transported back to the laboratory at room temperature. Our procedure allows the typing of several
molecular genetic markers from just a drop of blood collected in the field and stored at room temperature alleviating the need for storage
in expensive deep freezers or liquid nitrogen tanks.

Matched MeSH terms: DNA Primers
Fulltext Risk of Escherichia coli O157:H7 infection linked to the consumption of beef

Premarathne J.M.K.J.K., New, C.Y., Ubong, A, Nakaguchi, Y., Nishibuchi, M., Son, R.

Food Research, 2017;1(3):67-76.
MyJurnal

Escherichia coli O157:H7 is a major food-borne pathogen that has resulted in numerous
outbreaks around the world. Widespread distribution of the organism in various ecological
niches impedes the control measures. This study aimed to detect and quantify E. coli O157:H7
in beef sold in wet markets and hypermarkets in Malaysia and to determine the risk of E. coli
O157:H7 infection linked to consumption of beef. The rfbO157 and flicH7 primers targeted on
somatic antigen (O157) and flagellar antigen (H7) respectively of E. coli O157:H7 was used for
the MPN-PCR method. A total of 99 beef samples were collected from local wet markets and
hypermarkets. The highest E. coli O157:H7 contamination rate was observed in beef samples
collected from wet markets (89.50%), whereas the contamination rate in hyper market A and B
were compratively low (35.35 and 20% respectively). However, the microbial load was highest
in the beef samples from hypermarket A (1100 MPN/g) while E. coli O157:H7 bacterial load
in beef samples from hypermarket B and wet market ranged from 3 to 93 MPN/g and 3 to 240
MPN/g, respectively. Using the Quantitative Microbial Risk Assessment (QMRA) approach
the risk was estimated incorporating the findings of the prevalence study and predictions
based on home storage, cooking and consumption patterns. Three different exposure pathways
were investigated to estimate the risk associated with contaminated beef and Monte Carlo
simulation was used to determine the level of uncertainty. The developed model predicated that
consumption of contaminated beef can be accountable for 1.83E+06 E. coli O157:H7 cases per
year in Malaysia. The reliability of the model, data gaps and further research needs, is discussed.
Through continuous improvement Quantitative Microbial Risk Assessment provides valuable
insight into controlling and prevention strategies.

Matched MeSH terms: DNA Primers
Molecular Characterization of Begomoviruses Associated with Leafcurl Diseases of Tomato in Bangladesh, Laos, Malaysia, Myanmar, and Vietnam

Green SK, Tsai WS, Shih SL, Black LL, Rezaian A, Rashid MH, et al.

Plant Dis, 2001 Dec;85(12):1286.
PMID: 30831796 DOI: 10.1094/PDIS.2001.85.12.1286A

Production of tomato (Lycopersicon esculentum) in Bangladesh, Malaysia, Myanmar, Vietnam, and Laos has been severely affected by yellow leaf curl disease. Tomato leaf samples were collected from symptomatic tomato plants from farmers' fields in the five countries from 1997 to 1999. DNA was extracted from all samples, four from Vietnam, two each from Malaysia, Laos, and Myanmar, and seven from Bangladesh. Virus DNA was amplified by polymerase chain reaction (PCR) using the begomovirus-specific degenerate primer pair PAL1v 1978/PAR1c 715(1), which amplifies the top part of DNA A. All samples gave the expected 1.4-kb PCR product. The PCR product of one sample per country was cloned and sequenced. Based on the sequences of the 1.4-kb DNA products amplified by the first primer pair, specific primers were designed to complete each of the DNA A sequences. Computer-assisted sequence comparisons were performed with begomovirus sequences available in the laboratory at the Asian Vegetable Research and Development Center, Shanhua, Tainan, and in the GenBank sequence database. The five DNA species resembled DNA A of begomoviruses. For the detection of DNA B two degenerate primer pairs were used, DNABLC1/DNABLV2 and DNABLC2/DNABLV2 (DNABLC1: 5'-GTVAATGGRGTDCACTTCTG-3', DNABLC2: 5'-RGTDCACTT CTGYARGATGC-3', DNABLV2: 5'-GAGTAGTAGTGBAKGTTGCA-3'), which were specifically designed to amplify DNA B of Asian tomato geminiviruses. Only the virus associated with yellow leaf curl of tomato in Bangladesh was found to contain a DNA B component, which was detected with the DNABLC1/DNABLV2 primer pair. The DNA A sequence derived from the virus associated with tomato yellow leaf curl from Myanmar (GenBank Accession No. AF206674) showed highest sequence identity (94%) with tomato yellow leaf curl virus from Thailand (GenBank Accession No. X63015), suggesting that it is a closely related strain of this virus. The other four viruses were distinct begomoviruses, because their sequences shared less than 90% identity with known begomoviruses of tomato or other crops. The sequence derived from the virus associated with tomato yellow leaf curl from Vietnam (GenBank Accession No. AF264063) showed highest sequence identity (82%) with the virus associated with chili leaf curl from Malaysia (GenBank Accession No. AF414287), whereas the virus associated with yellow leaf curl symptoms in tomato in Bangladesh (GenBank Accession No. AF188481) had the highest sequence identity (88%) with a tobacco geminivirus from Yunnan, China (GenBank Accession No. AF240675). The sequence derived from the virus associated with tomato yellow leaf curl from Laos (GenBank Accession No. AF195782) had the highest sequence identity (88%) with the tomato begomovirus from Malaysia (GenBank Accession No. AF327436). This report provides further evidence of the great genetic diversity of tomato-infecting begomoviruses in Asia. Reference: M. R. Rojas et al. Plant Dis. 77:340, 1993.

Matched MeSH terms: DNA Primers
First Report of Tomato Gray Leaf Spot Disease Caused by Stemphylium solani in Malaysia

Nasehi A, Kadir JB, Abidin MAZ, Wong MY, Mahmodi F

Plant Dis, 2012 Aug;96(8):1226.
PMID: 30727066 DOI: 10.1094/PDIS-03-12-0223-PDN

In June 2011, tomatoes (Solanum lycopersicum) in major growing areas of the Cameron Highlands and the Johor state in Malaysia were affected by a leaf spot disease. Disease incidence exceeded 80% in some severely infected regions. Symptoms on 50 observed plants initially appeared on leaves as small, brownish black specks, which later became grayish brown, angular lesions surrounded by a yellow border. As the lesions matured, the affected leaves dried up and became brittle and later developed cracks in the center of the lesions. A survey was performed in these growing areas and 27 isolates of the pathogen were isolated from the tomato leaves on potato carrot agar (PCA). The isolates were purified by the single spore technique and were transferred onto PCA and V8 agar media for conidiophore and conidia production under alternating light (8 hours per day) and darkness (16 hours per day) (4). Colonies on PCA and V8 agar exhibited grey mycelium and numerous conidia were formed at the terminal end of conidiophores. The conidiophores were up to 240 μm long. Conidia were oblong with 2 to 11 transverse and 1 to 6 longitudinal septa and were 24 to 69.6 μm long × 9.6 to 14.4 μm wide. The pathogen was identified as Stemphylium solani on the basis of morphological criteria (2). In addition, DNA was extracted and the internal transcribed spacer region (ITS) was amplified by universal primers ITS5 and ITS4 (1). The PCR product was purified by the commercial PCR purification kit and the purified PCR product sequenced. The resulting sequences were 100% identical to published S. solani sequences (GenBank Accestion Nos. AF203451 and HQ840713). The amplified ITS region was deposited with NCBI GenBank under Accession No. JQ657726. A representative isolate of the pathogen was inoculated on detached 45-day-old tomato leaves of Malaysian cultivar 152177-A for pathogenicity testing. One wounded and two nonwounded leaflets per leaf were used in this experiment. The leaves were wounded by applying pressure to leaf blades with the serrated edge of a forceps. A 20-μl drop of conidial suspension containing 105 conidia/ml was used to inoculate these leaves (3). The inoculated leaves were placed on moist filter paper in petri dishes and incubated for 48 h at 25°C. Control leaves were inoculated with sterilized distilled water. After 7 days, typical symptoms for S. solani similar to those observed in the farmers' fields developed on both wounded and nonwounded inoculated leaves, but not on noninoculated controls, and S. solani was consistently reisolated. To our knowledge, this is the first report of S. solani causing gray leaf spot of tomato in Malaysia. References: (1) M. P. S. Camara et al. Mycologia 94:660, 2002. (2) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (3) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002. (4) E. G. Simmons. CBS Biodiversity Series 6:775, 2007.

Matched MeSH terms: DNA Primers
Prevalence of salmonella sp. In African catfish (clarias gariepinus) obtained from farms and wet markets in Kelantan, Malaysia and their antibiotic resistance

Sing CK, Md. Zahirul Islam Khan, Hassan Hj. Mohd Daud, Abd. Rahman Aziz

Sains Malaysiana, 2016;45:1597-1602.

The present study was conducted to determine the prevalence and antibiotic resistance of Salmonella sp. isolated from
African catfish (Clarias gariepinus). A total of 30 catfish were harvested from four different farms and four different
wet markets. A total of 60 samples (30 catfish skins and 30 catfish intestines) were used for Salmonella sp. isolation
(pellet-method), its biochemical and serological test. Confirmation of Salmonella sp. were determined by polyvalent
O antisera and polymerase chain reaction (PCR) using genus specific primers for invA genes (DNA amplification
showed one distinct band with molecular weight of 389 bp) and the species of isolated Salmonella sp. were identified
by serotyping. The result showed 6/30 (20%) of fish or 6/60 (10%) of organ samples were positive for Salmonella sp.
Among those positive for Salmonella sp., 4/6 were from intestine samples and 2/6 were from skin samples. No significant
difference was found in the prevalence of Salmonella sp. isolates between fish harvested from farms and wet markets
(p-value= 0.406). The Salmonella serovars identified were Salmonella corvallis (n=3), Salmonella mbandaka (n=2)
and Salmonella typhmurium (n=1). Salmonella sp. isolates were resistance to Penicillin (P 10, 100%), Clindamycin
(DA 2, 100%), Tetracycline (TE 30, 100%) and Rifampicin (RD 5, 100%) and all of the isolates were susceptible or
intermediate resistance to Ceftazidime (CAZ 30) and Trimethopin (W 5). Multiple antibiotic resistance (MAR) index of
all Salmonella sp. isolates in current study was 0.67 indicating that fish sampled in the present study was under high
risk of been exposed to the tested antibiotics.

Matched MeSH terms: DNA Primers

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