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Fulltext Effect of perivitelline fluid from horseshoe crab on the expression of COL1A1 in dental pulp stem cells

Amanina Fatinah Kamarudin, Najian Ibrahim, Thirumulu Ponnuraj Kannan, Ahmad Aizat Abdul Aziz

Archives of Orofacial Sciences, 2016;11(2):26-30.
MyJurnal

Perivitelline fluid, extracted from the fertilized eggs of horseshoe crabs, has been reported to play a
vital role in supporting embryogenesis as well as cell proliferation. The present study aims to evaluate the effect
of PVF on the expression of COL1A1 in human dental pulp stem cells (DPSCs). The cells were grouped into two;
untreated (control) and treated with a single dose of PVF (0.019 mg/ml). Gene expression was quantified for
COL1A1 on day 1, 3 and 7 using reverse transcriptase PCR. The expression of COL1A1 on day 3 of treated
group with PVF was the highest though there was a decline of COL1A1 expression on day 7. Mann Whitney test
was utilized to determine the significance of COL1A1 expression between treated and untreated groups.
Significant difference in the expression of COL1A1 was observed between the treated and untreated groups on
day 3 though there was no significance in the expression on day 7. The present study indicates that PVF may
have the potential to increase cell proliferation in human DPSCs.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext A simple and inexpensive physical lysis method for DNA and RNA extraction from freshwater microalgae

Yee W, Abdul-Kadir R, Lee LM, Koh B, Lee YS, Chan HY

3 Biotech, 2018 Aug;8(8):354.
PMID: 30105179 DOI: 10.1007/s13205-018-1381-1

In this work, a simple and inexpensive physical lysis method using a cordless drill fitted with a plastic pellet pestle and 150 mg of sterile sea sand was established for the extraction of DNA from six strains of freshwater microalgae. This lysis method was also tested for RNA extraction from two microalgal strains. Lysis duration between 15 and 120 s using the cetyltrimethyl ammonium bromide (CTAB) buffer significantly increased the yield of DNA from four microalgalstrains (Monoraphidium griffithii NS16, Scenedesmus sp. NS6, Scenedesmus sp. DPBC1 and Acutodesmus sp. DPBB10) compared to control. It was also found that grinding was not required to obtain DNA from two strains of microalgae (Choricystis sp. NPA14 and Chlamydomonas sp. BM3). The average DNA yield obtained using this lysis method was between 62.5 and 78.9 ng/mg for M. griffithii NS16, 42.2-247.0 ng/mg for Scenedesmus sp. NS6, 70.2-110.9 ng/mg for Scenedesmus sp. DPBC1 and 142.8-164.8 ng/mg for Acutodesmus sp. DPBB10. DNA obtained using this method was sufficiently pure for PCR amplification. Extraction of total RNA from M. griffithii NS16 and Mychonastes sp. NPD7 using this lysis method yielded high-quality RNA suitable for RT-PCR. This lysis method is simple, cheap and would enable rapid nucleic acid extraction from freshwater microalgae without requiring costly materials and equipment such as liquid nitrogen or beadbeaters, and would facilitate molecular studies on microalgae in general.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Selective inhibition of genes in methicillin resistant Staphylococcus aureus (MRSA) treated with Melastoma malabathricum methanol extract

Zulaikah Mohamed, Nazlina Ibrahim, Ismail Ahmad

Sains Malaysiana, 2008;37(1):107-113.

Methanol extract of Melastoma malabathricum leaves inhibited the growth of Staphylococcus aureus and six clinical isolates of Methicilin Resistant Stapyhlococcus aureus (MRSA 1-6). The minimum inhibitory concentration (MIC) of test substance was 1.565mg/ml and the minimum bacteriocidal concentration (MBC) was 3.125 mg/ml. The methanol extract suppressed RNA synthesis at 10 mg/ml as shown by RNA profile which was devoid of three bands compared to the control. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using seven primer pairs was only successful in amplifying four cDNA amplicons. The failure to amplify three cDNA amplicons for three primer pairs corresponding to gyrA, femA and nuc genes, implied the possibility of suppression of the corresponding mRNA. Electrophoretic separation of endogenous and exogenuos bacterial proteins showed that three and five protein, respectively were not expressed. One endogenous and three exogenous proteins were over-expressed in treated MRSA compared with untreated control. The results of the molecular and proteomic analyses are in agreement, and based on primers being used, methanol extract of M. malabathricum leaves possibly inhibits MRSA growth through inhibition of DNA synthesis, peptidoglycan production, and nuclease production.
Keywords: Methicillin resistant Staphylococcus aureus; Melastoma malabathricum; gene expression; protein production

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Detection of Sugarcane yellow leaf virus in Quarantine and Production of Virus-free Sugarcane by Apical Meristem Culture

Chatenet M, Delage C, Ripolles M, Irey M, Lockhart BEL, Rott P

Plant Dis, 2001 Nov;85(11):1177-1180.
PMID: 30823163 DOI: 10.1094/PDIS.2001.85.11.1177

Sugarcane yellow leaf virus (SCYLV) was detected for the first time in 1996 in the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) sugarcane quarantine at Montpellier by reverse transcription-polymerase chain reaction (RT-PCR) in varieties from Brazil, Florida, Mauritius, and Réunion. Between 1997 and 2000, the virus was found by RT-PCR and/or tissue-blot immunoassay (TBIA) in additional varieties from Barbados, Cuba, Guadeloupe, Indonesia, Malaysia, Philippines, Puerto Rico, and Taiwan, suggesting a worldwide distribution of the pathogen. An excellent correlation was observed between results obtained for the two diagnostic techniques. However, even though only a few false negative results were obtained by either technique, both are now used to detect SCYLV in CIRAD's sugarcane quarantine in Montpellier. The pathogen was detected by TBIA or RT-PCR in all leaves of sugarcane foliage, but the highest percentage of infected vascular bundles was found in the top leaves. The long hot water treatment (soaking of cuttings in water at 25°C for 2 days and then at 50°C for 3 h) was ineffective in eliminating SCYLV from infected plants. Sugarcane varieties from various origins were grown in vitro by apical bud culture and apical meristem culture, and the latter proved to be the most effective method for producing SCYLV-free plants.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Performance Evaluation of Commercial Dengue Diagnostic Tests for Early Detection of Dengue in Clinical Samples

Mat Jusoh TNA, Shueb RH

J Trop Med, 2017;2017:4687182.
PMID: 29379526 DOI: 10.1155/2017/4687182

The shattering rise in dengue virus infections globally has created a need for an accurate and validated rapid diagnostic test for this virus. Rapid diagnostic test (RDT) and reverse transcription-polymerase chain reaction (RT-PCR) diagnostic detection are useful tools for diagnosis of early dengue infection. We prospectively evaluated the diagnostic performance of nonstructural 1 (NS1) RDT and real-time RT-PCR diagnostic kits in 86 patient serum samples. Thirty-six samples were positive for dengue NS1 antigen while the remaining 50 were negative when tested with enzyme-linked immunosorbent assay (ELISA). Commercially available RDTs for NS1 detection, RTK ProDetect™, and SD Bioline showed high sensitivity of 94% and 89%, respectively, compared with ELISA. GenoAmp® Trioplex Real-Time RT-PCR and RealStar® Dengue RT-PCR tests presented a comparable kappa agreement with 0.722. The result obtained from GenoAmp® Real-Time RT-PCR Dengue test showed that 14 samples harbored dengue virus type 1 (DENV-1), 8 samples harbored DENV-2, 2 samples harbored DENV-3, and 1 sample harbored DENV-4. 1 sample had a double infection with DENV-1 and DENV-2. The NS1 RDTs and real-time RT-PCR tests were found to be a useful diagnostic for early and rapid diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext In Vitro Pro-apoptotic and Anti-migratory Effects of Ficus deltoidea L. Plant Extracts on the Human Prostate Cancer Cell Lines PC3

Hanafi MMM, Afzan A, Yaakob H, Aziz R, Sarmidi MR, Wolfender JL, et al.

Front Pharmacol, 2017;8:895.
PMID: 29326585 DOI: 10.3389/fphar.2017.00895

This study aims to evaluate the in vitro cytotoxic and anti-migratory effects of Ficus deltoidea L. on prostate cancer cells, identify the active compound/s and characterize their mechanism of actions. Two farmed varieties were studied, var. angustifolia (FD1) and var. deltoidea (FD2). Their crude methanolic extracts were partitioned into n-hexane (FD1h, FD2h) chloroform (FD1c, FD2c) and aqueous extracts (FD1a, FD2a). Antiproliferative fractions (IC50 < 30 μg/mL, SRB staining of PC3 cells) were further fractionated. Active compound/s were dereplicated using spectroscopic methods. In vitro mechanistic studies on PC3 and/or LNCaP cells included: annexin V-FITC staining, MMP depolarization measurements, activity of caspases 3 and 7, nuclear DNA fragmentation and cell cycle analysis, modulation of Bax, Bcl-2, Smac/Diablo, and Alox-5 mRNA gene expression by RT-PCR. Effects of cytotoxic fractions on 2D migration and 3D invasion were tested by exclusion assays and modified Boyden chamber, respectively. Their mechanisms of action on these tests were further studied by measuring the expression VEGF-A, CXCR4, and CXCL12 in PC3 cells by RT-PCR. FD1c and FD2c extracts induced cell death (P < 0.05) via apoptosis as evidenced by nuclear DNA fragmentation. This was accompanied by an increase in MMP depolarization (P < 0.05), activation of caspases 3 and 7 (P < 0.05) in both PC3 and LNCaP cell lines. All active plant extracts up-regulated Bax and Smac/DIABLO, down-regulated Bcl-2 (P < 0.05). Both FD1c and FD2c were not cytotoxic against normal human fibroblast cells (HDFa) at the tested concentrations. Both plant extracts inhibited both migration and invasion of PC3 cells (P < 0.05). These effects were accompanied by down-regulation of both VEGF-A and CXCL-12 gene expressions (P < 0.001). LC-MS dereplication using taxonomy filters and molecular networking databases identified isovitexin in FD1c; and oleanolic acid, moretenol, betulin, lupenone, and lupeol in FD2c. In conclusion, FD1c and FD2c were able to overcome three main hallmarks of cancer in PC3 cells: (1) apoptosis by activating of the intrinsic pathway, (2) inhibition of both migration and invasion by modulating the CXCL12-CXCR4 axis, and (3) inhibiting angiogenesis by modulating VEGF-A expression. Moreover, isovitexin is here reported for the first time as an antiproliferative principle (IC50 = 43 μg/mL, SRB staining of PC3 cells).

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Development of a TaqMan minor groove binding probe-based quantitative reverse transcription polymerase chain reaction for the detection and quantification of Zika virus

Chin KL, Teoh BT, Sam SS, Loong SK, Tan KK, Azizan NS, et al.

Trop Biomed, 2022 Dec 01;39(4):518-523.
PMID: 36602210 DOI: 10.47665/tb.39.4.005

Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Detection of beta-globin gene mutations among Kelantan Malay thalassaemia patients by polymerase chain reaction restriction fragment length polymorphism

Rozitah R, Nizam MZ, Nur Shafawati AR, Nor Atifah MA, Dewi M, Kannan TP, et al.

Singapore Med J, 2008 Dec;49(12):1046-9.
PMID: 19122960

Beta-thalassaemia major is an autosomal recessive disorder that results in severe microcytic, hypochromic, haemolytic anaemia among affected patients. Beta-thalassaemia has emerged as one of the most common public health problems in Malaysia, particularly among Malaysian Chinese and Malays. This study aimed to observe the spectrum of mutations found in Kelantan Malay beta-thalassaemia major patients who attended the Paediatrics Daycare Unit, Hospital Universiti Sains Malaysia, Kelantan, Malaysia, the data of which was being used in establishing the prenatal diagnosis in this Human Genome Centre.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Performance evaluation of two multiplex qualitative RT-PCR assays for detection of respiratory infection in paediatric population

Ali U, Zainal M, Zainol Z, Tai CW, Tang SF, Lee PC, et al.

Malays J Pathol, 2023 Aug;45(2):215-227.
PMID: 37658531

INTRODUCTION: Acute respiratory infection (ARI) contributes to significant mortality and morbidity worldwide and is usually caused by a wide range of respiratory pathogens. This study aims to describe the performance of QIAstat-Dx® Respiratory Panel V2 (RP) and RespiFinder® 2SMART assays for respiratory pathogens detection.
MATERIALS AND METHODS: A total of 110 nasopharyngeal swabs (NPS) were collected from children aged one month to 12 years old who were admitted with ARI in UKMMC during a one-year period. The two qPCR assays were conducted in parallel.
RESULTS: Ninety-seven samples (88.2%) were positive by QIAstat-Dx RP and 86 (78.2%) by RespiFinder assay. The overall agreement on both assays was substantial (kappa value: 0.769) with excellent concordance rate of 96.95%. Using both assays, hRV/EV, INF A/H1N1 and RSV were the most common pathogens detected. Influenza A/H1N1 infection was significantly seen higher in older children (age group > 60 months old) (53.3%, p-value < 0.05). Meanwhile, RSV and hRV/EV infection were seen among below one-year-old children. Co-infections by two to four pathogens were detected in 17 (17.5%) samples by QIAstat-Dx RP and 12 (14%) samples by RespiFinder, mainly involving hRV/EV. Bacterial detection was observed only in 5 (4.5%) and 6 (5.4%) samples by QIAstat-Dx RP and RespiFinder, respectively, with Mycoplasma pneumoniae the most common detected.
CONCLUSION: The overall performance of the two qPCR assays was comparable and showed excellent agreement. Both detected various clinically important respiratory pathogens in a single test with simultaneous multiple infection detection. The use of qPCR as a routine diagnostic test can improve diagnosis and management.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
The mazEF toxin-antitoxin system as a novel antibacterial target in Acinetobacter baumannii

Ghafourian S, Good L, Sekawi Z, Hamat RA, Soheili S, Sadeghifard N, et al.

Mem Inst Oswaldo Cruz, 2014 Jul;109(4):502-5.
PMID: 25004148

Although analysis of toxin-antitoxin (TA) systems can be instructive, to date, there is no information on the prevalence and identity of TA systems based on a large panel of Acinetobacter baumannii clinical isolates. The aim of the current study was to screen for functional TA systems among clinical isolates of A. baumannii and to identify the systems' locations. For this purpose, we screened 85 A. baumannii isolates collected from different clinical sources for the presence of the mazEF, relBE and higBA TA genes. The results revealed that the genes coding for the mazEF TA system were commonly present in all clinical isolates of A. baumannii. Reverse transcriptase-polymerase chain reaction analysis showed that transcripts were produced in the clinical isolates. Our findings showed that TA genes are prevalent, harboured by chromosomes and transcribed within A. baumannii. Hence, activation of the toxin proteins in the mazEF TA system should be investigated further as an effective antibacterial strategy against this bacterium.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Gene expression profiles in human HepG2 cells treated with extracts of the Tamarindus indica fruit pulp

Razali N, Aziz AA, Junit SM

Genes Nutr, 2010 Dec;5(4):331-41.
PMID: 21189869 DOI: 10.1007/s12263-010-0187-5

Tamarindus indicaL. (T. indica) or locally known as asam jawa belongs to the family of Leguminosae. The fruit pulp had been reported to have antioxidant activities and possess hypolipidaemic effects. In this study, we attempted to investigate the gene expression patterns in human hepatoma HepG2 cell line in response to treatment with low concentration of the fruit pulp extracts. Microarray analysis using Affymetrix Human Genome 1.0 S.T arrays was used in the study. Microarray data were validated using semi-quantitative RT-PCR and real-time RT-PCR. Amongst the significantly up-regulated genes were those that code for the metallothioneins (MT1M, MT1F, MT1X) and glutathione S-transferases (GSTA1, GSTA2, GST02) that are involved in stress response. APOA4, APOA5, ABCG5 and MTTP genes were also significantly regulated that could be linked to hypolipidaemic activities of the T. indica fruit pulp.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People's Republic of China

Li L, He Y, Yang H, Zhu J, Xu X, Dong J, et al.

J Clin Microbiol, 2005 Aug;43(8):3835-9.
PMID: 16081920

The genetic and phylogenetic characteristics of human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) sampled from children with hand, foot, and mouth disease in Shenzhen, People's Republic of China, over a 6-year period (1999 to 2004) were examined with reverse transcription-PCR and DNA sequencing. Out of 147 stool specimens, 60 showed positive signals when screened with EV71- and CA16-specific primers. EV71 was identified in 19 specimens, and CA16 was identified in 41 specimens; coinfection by EV71 and CA16 was not observed. Phylogenetic analysis of all EV71 strains isolated from the mainland Chinese samples established C4 as the predominant genotype. Only one other known strain (3254-TAI-98; AF286531), isolated in Taiwan in 1998, was identified as belonging to genotype C4. Phylogenetic analysis of CA16 strains allowed us to identify three new genetic lineages (A, B, and C), with lineage C recently predominating in Asian countries, such as the People's Republic of China, Malaysia, and Japan. These new observations indicate that CA16 circulating in the People's Republic of China is genetically diverse, and additional surveillance is warranted.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Gelam (Melaleuca spp.) Honey-Based Hydrogel as Burn Wound Dressing

Mohd Zohdi R, Abu Bakar Zakaria Z, Yusof N, Mohamed Mustapha N, Abdullah MN

Evid Based Complement Alternat Med, 2012;2012:843025.
PMID: 21941590 DOI: 10.1155/2012/843025

A novel cross-linked honey hydrogel dressing was developed by incorporating Malaysian honey into hydrogel dressing formulation, cross-linked and sterilized using electron beam irradiation (25 kGy). In this study, the physical properties of the prepared honey hydrogel and its wound healing efficacy on deep partial thickness burn wounds in rats were assessed. Skin samples were taken at 7, 14, 21, and 28 days after burn for histopathological and molecular evaluations. Application of honey hydrogel dressings significantly enhanced (P < 0.05) wound closure and accelerated the rate of re-epithelialization as compared to control hydrogel and OpSite film dressing. A significant decrease in inflammatory response was observed in honey hydrogel treated wounds as early as 7 days after burn (P < 0.05). Semiquantitative analysis using RT-PCR revealed that treatment with honey hydrogel significantly (P < 0.05) suppressed the expression of proinflammatory cytokines (IL-1α, IL-1β, and IL-6). The present study substantiates the potential efficacy of honey hydrogel dressings in accelerating burn wound healing.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext In Vivo Antioxidant and Hypolipidemic Effects of Fermented Mung Bean on Hypercholesterolemic Mice

Yeap SK, Beh BK, Ho WY, Mohd Yusof H, Mohamad NE, Ali NM, et al.

Evid Based Complement Alternat Med, 2015;2015:508029.
PMID: 26074993 DOI: 10.1155/2015/508029

Legumes have previously been reported with hypolipidemic effect caused by the presence of flavonoid. This study was carried out to evaluate the antioxidant and hypolipidemic effects of fermented mung bean on hypercholesterolemic mice. Blood from all mice was collected and subjected to serum lipid and liver profiles biochemical analysis and quantitative RT-PCR for atherosclerosis related gene expressions. Besides, livers were collected for antioxidant assays and histopathology evaluation. Fermented mung bean was found to reduce the level of serum lipid and liver enzyme profiles of hypercholesterolemic mice. Furthermore, liver antioxidant and nitric oxide levels were also significantly restored by fermented mung bean in a dosage dependent manner. The gene expression study indicated that Apoe and Bcl2a1a were upregulated while Npy and Vwf expressions were downregulated after the treatment. The effects of fermented mung bean were greater than nonfermented mung bean. These results indicated that fermented mung bean possessed antioxidants that lead to its hypolipidemic effect on hypercholesterolemic mice.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Polymerase chain reaction-based discrimination of viable from non-viable Mycoplasma gallisepticum

Tan CG, Ideris A, Omar AR, Yii CP, Kleven SH

Onderstepoort J Vet Res, 2014 09 02;81(1):e1-e7.
PMID: 25686255 DOI: 10.4102/ojvr.v81i1.708

The present study was based on the reverse transcription polymerase chain reaction (RT-PCR) of the 16S ribosomal nucleic acid (rRNA) of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20-25 h at 37 °C, 22-25 h at 16 °C, and 23-27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h). The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Fulltext A comparative evaluation of dengue diagnostic tests based on single-acute serum samples for laboratory confirmation of acute dengue

Chua KB, Mustafa B, Abdul Wahab AH, Chem YK, Khairul AH, Kumarasamy V, et al.

Malays J Pathol, 2011 Jun;33(1):13-20.
PMID: 21874746

A prospective study was carried out to evaluate the sensitivity of dengue NS1 antigen-capture ELISA in comparison with dengue virus isolation, conventional RT-PCR and real-time RT-PCR for laboratory confirmation of acute dengue based on single-acute serum samples. Four primary healthcare centres were involved to recruit patients with clinical diagnosis of dengue illness. Patient's demographic, epidemiological and clinical information were collected on a standardized data entry form and 5 ml of venous blood was collected upon consent. In the laboratory, six types of laboratory tests were performed on each of the collected acute serum sample. Of the 558 acute serum samples collected from 558 patients with clinical diagnosis of dengue from mid-August 2006 to March 2009, 174 serum samples were tested positive by the dengue NS1 antigen-capture ELISA, 77 by virus isolation, 92 by RT-PCR and 112 by real-time RT-PCR. A total of 190 serum samples were tested positive by either one or a combination of the four methods whereas, only 59 serum samples were tested positive by all four methods. Thus, based on single-acute serum samples, 190 of the 558 patients (34.1%) were laboratory-confirmed acute dengue. The overall test sensitivity was 91.6%, 40.5%, 48.4% and 58.9% for dengue NS1 antigen-capture ELISA, virus isolation, conventional RT-PCR and real-time RT-PCR respectively. Statistically, dengue NS1 antigen-capture ELISA was the most sensitive and virus isolation was the least sensitive test for the laboratory confirmation of acute dengue based on single-acute serum specimens. Real-time RT-PCR was significantly more sensitive than the conventional RT-PCR.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
A modified in situ RT-PCR method for localizing fungal-specific gene expression in Candida-infected mice renal cells

Yong VC, Ong KW, Sidik SM, Rosli R, Chong PP

J Microbiol Methods, 2009 Nov;79(2):242-5.
PMID: 19737582 DOI: 10.1016/j.mimet.2009.08.019

In situ Reverse Transcriptase PCR (in situ RT-PCR) can amplify mRNA and localize gene expression in cells. However, this method is not feasible in fungi as the thick fungal cell wall constitutes a barrier to this procedure. We developed a two step in situ RT-PCR procedure which enabled the detection and localization of Candida tropicalis mRNA expression in formalin-fixed, paraffin-embedded (FFPE) mouse kidney sections. This in situ hybridization study revealed the first direct evidence for deposition of Candida tropicalis secreted aspartic proteinase 2 (CtSAP2) in the tip of pseudohyphae and its involvement in acute systemic candidiasis. We conclude that in situ RT-PCR can be successfully applied to FFPE tissues and will offer new perspectives in studying gene expression in Candida species.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Molecular detection of enteroviruses from an outbreak of hand, foot and mouth disease in Malaysia in 1997

Abubakar S, Chee HY, Shafee N, Chua KB, Lam SK

Scand. J. Infect. Dis., 1999;31(4):331-5.
PMID: 10528868

Enterovirus 5'UTR sequences were detected by RT-PCR in 22 out of 47 suspected hand, foot and mouth disease (HFMD) patients during an outbreak of the disease with incidences of fatal brainstem encephalomyelitis in Malaysia in 1997. Genetic and phylogenetic analyses of the isolates 5'UTR sequences suggest the presence of predominantly enteroviruses with high sequence similarities to Echovirus 1 and Coxsackievirus A9 in the Malaysian peninsula. No fatal cases, however, were associated with these isolates. The remaining isolates, including all (4/4) isolates of the fatal cases from the Malaysian peninsula and Sarawak shared very high sequence identity with enterovirus 71MS (EV71). These findings suggest that several enteroviruses were circulating in Malaysia during the outbreak period, with only EV71 causing fatal infections.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction*
Sequence analysis of Malaysian infectious bursal disease virus isolate and the use of reverse transcriptase nested polymerase chain reaction enzyme-linked immunosorbent assay for the detection of VP2 hypervariable region

Phong SF, Hair-Bejo M, Omar AR, Aini I

Avian Dis, 2003 Jan-Mar;47(1):154-62.
PMID: 12713171

The VP2 hypervariable region of P97/302 local infectious bursal disease virus (IBDV) isolate was amplified by the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and cloned. This region of P97/302 local isolate was sequenced and compared with eight other reported IBDV sequences. The result showed that P97/302 IBDV was most identical to the reported very virulent IBDV strains because it has amino acid substitutions at positions 222, 256, 294, and 299, which encode alanine, isoleucine, isoleucine, and serine, respectively. This region can be digested with restriction enzymes of Taq1, Sty1, Ssp1 but not with Sac1. The P97/302 isolate was then used for the optimization of RT nested PCR enzyme-linked immunosorbent assay (ELISA). The RT nested PCR ELISA was able to detect 10(-4) dilution of the infected bursa homogenates and was 10 times more sensitive when compared with the agarose gel detection method. The RT nested PCR ELISA can detect up to 0.48 ng of the PCR product. The specificity of this nested PCR ELISA was also high (100%).

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Molecular evidence and hematological alterations associated with the occurrence of coronavirus in domestic dogs in Pakistan

Sulehria MU, Ahmad SS, Ijaz M, Mushtaq MH, Khan AY, Ghaffar A

Trop Biomed, 2020 Dec 01;37(4):963-972.
PMID: 33612749 DOI: 10.47665/tb.37.4.963

Canine Enteric Coronavirus (CCoV) is one of the major enteric pathogen affecting dogs. This study aims to investigate the molecular prevalence, phylogenetic analysis, associated risk factors, and haemato-biochemical alterations in Canine Coronavirus in dogs in district Lahore, Pakistan. 450 fecal samples were collected from symptomatic dogs originating from various pet-clinics and kennels during 2018-2019. Samples were initially analyzed by sandwich lateral flow immunochromatographic assay and then further processed by RT-PCR (reverse transcriptase polymerase chain reaction) targeting the M gene followed by sequencing. RT-PCR based positive (n=20) and negative (n=20) dogs were samples for their blood for the haemato-biochemical analysis. A questionnaire was used to collect data from pet owners, in order to analyze the data for risk factors analysis by chi square test on SPSS. The prevalence of CCoV was 35.1%, and 23.8 % through Sandwich lateral flow immunochromatographic and RT-PCR respectively. Various risk factors like breed, age, sex, vomiting, diarrhea, sample source, body size, cohabitation with other animals, living environment, food, deworming history, contact with other animals or birds feces, and season were significantly associated with CCoV. The CCoV identified in Pakistan were 98% similar with the isolates from China (KT 192675, 1), South Korea (HM 130573, 1), Brazil (GU 300134, 1), Colombia (MH 717721, 1), United Kingdom (JX 082356, 1) and Tunisia (KX156806). Haematobiochemical alterations in CCoV affected dogs revealed anaemia, leucopenia, lymphopenia, neutrophilia, and decreased packed cell volume, and a significant increase in alkaline phosphate and alanine transaminase. It is concluded that infection with canine coronavirus appears widespread among dog populations in district Lahore, Pakistan. This study is the first report regarding the molecular detection and sequence analysis of CCoV in Pakistan.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/veterinary

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