MyMedR

Displaying publications 81 - 100 of 314 in total

Abstract:

Sort:

Plasmodium knowlesi: from Malaysia, a novel health care threat

Sabbatani S, Fiorino S, Manfredi R

Infez Med, 2012 Mar;20(1):5-11.
PMID: 22475654

Epidemic foci of Plasmodium knowlesi malaria have been identified during the past ten years in Malaysia, in particular in the States of Sarawak and Sabah (Malaysia Borneo), and in the Pahang region (peninsular Malaysia). Based on a review of the available recent international literature, the authors underline the importance of molecular biology examinations, polymerase chain reactions (PCR), performed with primers specific for P. knowlesi, since the current microscopic examination (haemoscope) may fail to distinguish P. knowlesi from Plasmodium malariae, due to the very similar appearance of the two parasites. P. knowlesi has been described as the causal agent of life-threatening and lethal forms of malaria: its clinical picture is more severe when compared with that of P. malariae, since the disease is characterized by greater parasitaemia, as opposed to that documented in the course of P. malariae disease. The most effective carrier is Anopheles leucosphyrus: this mosquito is attracted by both humans and monkeys. Among primates, the natural hosts of P. knowlesi are Macaca fascicularis and Macaca nemestina, while Saimiri scirea and Macaca mulatta, which cannot become infected in nature, may be useful in experimental models. When underlining the potentially severe evolution, we note the key role played by prompt disease recognition, which is expected to be more straightforward in patients monitored in endemic countries at high risk, but should be carefully implemented for subjects being admitted to hospital in Western countries suffering from the typical signs and symptoms of malaria, after travelling in South-East Asia where they were engaged in excursions in the tropical forest (trekking, and similar outdoor activities). In these cases, the diagnosis should be prompt, and suitable treatment should follow. According to data in the literature, in non-severe cases chloroquine proves very effective against P. knowlesi, achieving the disappearance of signs and symptoms in 96% of cases after only 24 hours after treatment start. In the light of the emerging epidemiological data, P. knowlesi should be added to Plasmodium vivax, Plasmodium ovale, P. malariae, and Plasmodium falciparum, as the fifth aetiological agent of malaria. During the next few years, it will become mandatory to plan an appropriate surveillance program of the epidemiological evolution, paying also great attention to the clinical features of patients affected by P. knowlesi malaria, which are expected to worsen according to the time elapsed; some studies seem to point out greater severity according to increased parasitaemia, paralleling the increased interhuman infectious passages of the plasmodium.

Matched MeSH terms: Polymerase Chain Reaction/methods
Fulltext Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening

Cox-Singh J, Davis TM, Lee KS, Shamsul SS, Matusop A, Ratnam S, et al.

Clin Infect Dis, 2008 Jan 15;46(2):165-71.
PMID: 18171245 DOI: 10.1086/524888

BACKGROUND: Until recently, Plasmodium knowlesi malaria in humans was misdiagnosed as Plasmodium malariae malaria. The objectives of the present study were to determine the geographic distribution of P. knowlesi malaria in the human population in Malaysia and to investigate 4 suspected fatal cases.
METHODS: Sensitive and specific nested polymerase chain reaction was used to identify all Plasmodium species present in (1) blood samples obtained from 960 patients with malaria who were hospitalized in Sarawak, Malaysian Borneo, during 2001-2006; (2) 54 P. malariae archival blood films from 15 districts in Sabah, Malaysian Borneo (during 2003-2005), and 4 districts in Pahang, Peninsular Malaysia (during 2004-2005); and (3) 4 patients whose suspected cause of death was P. knowlesi malaria. For the 4 latter cases, available clinical and laboratory data were reviewed.
RESULTS: P. knowlesi DNA was detected in 266 (27.7%) of 960 of the samples from Sarawak hospitals, 41 (83.7%) of 49 from Sabah, and all 5 from Pahang. Only P. knowlesi DNA was detected in archival blood films from the 4 patients who died. All were hyperparasitemic and developed marked hepatorenal dysfunction.
CONCLUSIONS: Human infection with P. knowlesi, commonly misidentified as the more benign P. malariae, are widely distributed across Malaysian Borneo and extend to Peninsular Malaysia. Because P. knowlesi replicates every 24 h, rapid diagnosis and prompt effective treatment are essential. In the absence of a specific routine diagnostic test for P. knowlesi malaria, we recommend that patients who reside in or have traveled to Southeast Asia and who have received a "P. malariae" hyperparasitemia diagnosis by microscopy receive intensive management as appropriate for severe falciparum malaria.

Matched MeSH terms: Polymerase Chain Reaction/methods
Fulltext Plasmodium knowlesi malaria in Vietnam: some clarifications

Van den Eede P, Vythilingam I, Ngo DT, Nguyen VH, Le XH, D'Alessandro U, et al.

Malar J, 2010;9:20.
PMID: 20082717 DOI: 10.1186/1475-2875-9-20

A recently published comment on a report of Plasmodium knowlesi infections in Vietnam states that this may not accurately represent the situation in the study area because the PCR primers used may cross-hybridize with Plasmodium vivax. Nevertheless, P. knowlesi infections have been confirmed by sequencing. In addition, a neighbour-joining tree based on the 18S S-Type SSUrRNA gene shows that the Vietnamese samples clearly cluster with the P. knowlesi isolates identified in Malaysia and are distinct from the corresponding P. vivax sequences. All samples came from asymptomatic individuals who did not consult for fever during the months preceding or following the survey, indicating that asymptomatic P. knowlesi infections occur in this population, although this does not exclude the occurrence of symptomatic cases. Large-scale studies to determine the extent and the epidemiology of P. knowlesi malaria in Vietnam are further needed.

Matched MeSH terms: Polymerase Chain Reaction/methods
Performance of an RT-nested PCR ELISA for detection of Newcastle disease virus

Kho CL, Mohd-Azmi ML, Arshad SS, Yusoff K

J Virol Methods, 2000 Apr;86(1):71-83.
PMID: 10713378

A sensitive and specific RT-nested PCR coupled with an ELISA detection system for detecting Newcastle disease virus is described. Two nested pairs of primer which were highly specific to all the three different pathotypes of NDV were designed from the consensus fusion gene sequence. No cross-reactions with other avian infectious agents such as infectious bronchitis virus, infectious bursal disease virus, influenza virus, and fowl pox virus were observed. Based on agarose electrophoresis detection, the RT-nested PCR was about 100 times more sensitive compared to that of a non-nested RT-PCR. To facilitate the detection of the PCR product, an ELISA detection method was then developed to detect the amplified PCR products and it was shown to be ten times more sensitive than gel electrophoresis. The efficacy of the nested PCR-ELISA was also compared with the conventional NDV detection method (HA test) and non-nested RT-PCR by testing against a total of 35 tissue specimens collected from ND-symptomatic chickens. The RT-nested PCR ELISA found NDV positive in 21 (60%) tissue specimens, while only eight (22.9%) and two (5.7%) out of 35 tissue specimens were tested NDV positive by both the non-nested RT-PCR and conventional HA test, respectively. Due to its high sensitivity for the detection of NDV from tissue specimens, this PCR-ELISA based diagnostic test may be useful for screening large number of samples.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Fulltext Performance of a Taqman Assay for Improved Detection and Quantification of Human Rhinovirus Viral Load

Ng KT, Chook JB, Oong XY, Chan YF, Chan KG, Hanafi NS, et al.

Sci Rep, 2016 10 10;6:34855.
PMID: 27721388 DOI: 10.1038/srep34855

Human rhinovirus (HRV) is the major aetiology of respiratory tract infections. HRV viral load assays are available but limitations that affect accurate quantification exist. We developed a one-step Taqman assay using oligonucleotides designed based on a comprehensive list of global HRV sequences. The new oligonucleotides targeting the 5'-UTR region showed high PCR efficiency (E = 99.6%, R2 = 0.996), with quantifiable viral load as low as 2 viral copies/μl. Assay evaluation using an External Quality Assessment (EQA) panel yielded a detection rate of 90%. When tested on 315 human enterovirus-positive specimens comprising at least 84 genetically distinct HRV types/serotypes (determined by the VP4/VP2 gene phylogenetic analysis), the assay detected all HRV species and types, as well as other non-polio enteroviruses. A commercial quantification kit, which failed to detect any of the EQA specimens, produced a detection rate of 13.3% (42/315) among the clinical specimens. Using the improved assay, we showed that HRV sheds in the upper respiratory tract for more than a week following acute infection. We also showed that HRV-C had a significantly higher viral load at 2-7 days after the onset of symptoms (p = 0.001). The availability of such assay is important to facilitate disease management, antiviral development, and infection control.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods; Real-Time Polymerase Chain Reaction/methods*
Fulltext Pentaplex PCR assay for detection of hemorrhagic bacteria from stool samples

Al-Talib H, Latif B, Mohd-Zain Z

J Clin Microbiol, 2014 Sep;52(9):3244-9.
PMID: 24958797 DOI: 10.1128/JCM.00891-14

Diarrheal diseases cause illness and death among children younger than 10 years in developing countries. Conventional testing for the detection of hemorrhagic bacteria takes 2 to 5 days to yield complete information on the organism and its antibiotic sensitivity pattern. Hence, in the present study, we developed a molecular-based diagnostic assay that identifies common hemorrhagic bacteria in stool samples. A set of specific primers were designed for the detection of Salmonella spp., Shigella spp., enterohemorrhagic Escherichia coli (EHEC), and Campylobacter spp., suitable for use in a one-tube PCR assay. The assay in the present study simultaneously detected five genes, namely, ompC for the Salmonella genus, virA for the Shigella genus, eaeA for EHEC, 16S rRNA for the Campylobacter genus, and hemA for an internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. Validation with 20 Gram-negative and 17 Gram-positive strains yielded 100% specificity. The limit of detection of the multiplex PCR assay was 1 × 10(3) CFU at the bacterial cell level and 100 pg at the genomic DNA level. Further evaluation of the multiplex PCR with 223 bacterium-spiked stool specimens revealed 100% sensitivity and specificity. We conclude that the developed multiplex PCR assay was rapid, giving results within 4 h, which is essential for the identification of hemorrhagic bacteria, and it might be useful as an additional diagnostic tool whenever time is important in the diagnosis of hemorrhagic bacteria that cause diarrhea. In addition, the presence of an internal control in the multiplex PCR assay is important for excluding false-negative cases.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
Pathogenic and saprophytic Leptospira species in water and soils from selected urban sites in peninsular Malaysia

Benacer D, Woh PY, Mohd Zain SN, Amran F, Thong KL

Microbes Environ, 2013;28(1):135-40.
PMID: 23363618

Leptospira species were studied in water and soils from selected urban sites in Malaysia. A total of 151 water (n=121) and soil (n=30) samples were collected from 12 recreational lakes and wet markets. All samples were filtered and inoculated into semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media supplemented with additional 5-fluorouracil. The cultures were then incubated at 30°C and observed under a dark field microscope with intervals of 10 days. A PCR assay targeting the rrs gene was used to confirm the genus Leptospira among the isolates. Subsequently, the pathogenic status of the isolates was determined using primer sets G1/G2 and Sapro1/Sapro2, which target the secY and rrs genes, respectively. The isolates were identified at serogroup level using the microscopic agglutination test (MAT) while their genetic diversity was assessed by pulsed field gel electrophoresis (PFGE). Based on dark field microscopy, 23.1% (28/121) water and 23.3% (7/30) soil cultures were positive for Leptospira spp. Of the 35 positive cultures, only 8 were pure and confirmed as Leptospira genus by PCR assay. Two out of 8 isolates were confirmed as pathogenic, 5 were saprophytic and one was intermediate. These 8 isolates were negative for the 25 reference hyperimmune rabbit sera tested in the MAT. PFGE showed that all 8 of these environmental Leptospira spp. were genetically diverse. In conclusion, the presence of pathogenic Leptospira spp. in the urban Malaysian environment may indicate and highlight the importance of water screening, especially in recreational lakes, in order to minimize any chance of Leptospira infection.

Matched MeSH terms: Polymerase Chain Reaction/methods
Pathogenesis and antibody response to a cytomegalovirus infection in newborn rats

Loh HS, Mohd-Azmi ML, Sheikh-Omar AR, Zamri-Saad M, Tam YJ

Acta Virol., 2007;51(1):27-33.
PMID: 17432941

The present study described the kinetics of Rat cytomegalovirus (RCMV) infection in newborn rats by monitoring infectious virus and viral antigens in various organs, viral DNA in the blood (DNAemia) and antibody response. These parameters were evaluated quantitatively using double-antibody sandwich ELISA (DAS-ELISA), real-time PCR, indirect ELISA and virus infectivity assay. For the first time DAS-ELISA was used for detection of RCMV antigen directly from organ samples. The relationships between the presence of viral antigens in the infected organs and antibody levels were established by the Spearman's rank test. It was found that the virus was present in the blood, spleen, liver, lungs, and kidneys earlier than in the salivary glands. Furthermore, the early immunity of the newborn rats led to a delayed seroconversion. We suggested that the prolonged presence of the virus in salivary glands could augment the antibody response that conversely might be responsible for a reduction of viremia. This study expanded our understanding of RCMV pathogenesis leading to improved therapeutic and preventive treatment regimens particularly for the neonatal Human cytomegalovirus (HCMV) infections. Additionally, the detection procedures developed in this study such as DAS-ELISA and real-time PCR could serve as alternative techniques for rapid screening of large number of samples.

Matched MeSH terms: Polymerase Chain Reaction/methods
PCR-ELISA for the detection of Brugia malayi infection using finger-prick blood

Rahmah N, Ashikin AN, Anuar AK, Ariff RH, Abdullah B, Chan GT, et al.

Trans R Soc Trop Med Hyg, 1998 12 16;92(4):404-6.
PMID: 9850392

A polymerase chain reaction assay based on the enzyme-linked immunosorbent assay (PCR-ELISA) has been developed to detect Brugia malayi infection in an area of low endemicity in Malaysia. Blood samples from 239 subjects were tested: 192 amicrofilaraemic individuals, 14 microfilaraemic persons and 3 chronic elephantiasis cases from endemic areas and 30 city-dwellers (non-endemic controls). PCR products were examined by ELISA and Southern hybridization. In the PCR-ELISA, digoxigenin-labelled PCR products were hybridized to a biotin-labelled probe. This was followed by incubation in streptavidin-coated microtitre wells and detection using anti-digoxigenin-peroxidase and ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)]. All microfilaraemic samples were positive by PCR-ELISA and Southern hybridization and all samples from non-endemic subjects and chronic elephantiasis patients were negative. The PCR-ELISA detected 12 times as many B. malayi infections as did thick blood film examination. Nineteen of the 194 samples from the endemic area gave positive results by both PCR-ELISA and Southern hybridization, and an additional 5 samples were positive by PCR-ELISA only. The PCR-ELISA was specific and sensitive, detected more infections, and was more reproducible than Southern hybridization.

Matched MeSH terms: Polymerase Chain Reaction/methods*
PCR tools for the verification of the specific identity of ascaridoid nematodes from dogs and cats

Li MW, Lin RQ, Chen HH, Sani RA, Song HQ, Zhu XQ

Mol Cell Probes, 2007 Oct-Dec;21(5-6):349-54.
PMID: 17532185

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of Toxocara canis, Toxocara cati, Toxocara malaysiensis and Toxascaris leonina, specific forward primers were designed in the ITS-1 or ITS-2 for each of the four ascaridoid species of dogs and cats. These primers were used individually together with a conserved primer in the large subunit of rDNA to amplify partial ITS-1 and/or ITS-2 of rDNA from 107 DNA samples from ascaridoids from dogs and cats in China, Australia, Malaysia, England and the Netherlands. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the sequences amplified. The minimum amounts of DNA detectable using the PCR assays were 0.13-0.54ng. These PCR assays should provide useful tools for the diagnosis and molecular epidemiological investigations of toxocariasis in humans and animals.

Matched MeSH terms: Polymerase Chain Reaction/methods*
PCR identification and phylogenetic analysis of the medically important dust mite Suidasia medanensis (Acari: Suidasiidae) in Malaysia

Ernieenor FCL, Ernna G, Jafson AS, Mariana A

Exp Appl Acarol, 2018 Sep;76(1):99-107.
PMID: 30151715 DOI: 10.1007/s10493-018-0285-4

The occurrence of Suidasia medanensis (= S. pontifica) mites in Malaysian house dust was first reported in 1984. The taxonomy of this storage mite is, however, quite confusing. Therefore, we need an accurate identification to resolve morphological problems due to its minute size and some overlapping characters between species. The purpose of this study was to demonstrate the application of partial mitochondrial cytochrome c oxidase subunit I (COI) sequences for the identification of S. medanensis by PCR. Identity of the mite was first determined by observing morphological characters under a light microscope. Genomic DNA of S. medanensis mites was successfully extracted prior to PCR and DNA sequencing using COI universal primers. The length of the COI sequences obtained was 378 bp. BLAST analysis of amplicon sequences showed that local S. medanensis COI region had 99% maximum identity with S. medanensis nucleotide sequence (AY525568) available in the GenBank. As the phylogenetic tree generated indicated, COI sequences from this study were clustered with S. medanensis from Korea and the UK in one major clade, supported with high bootstrap value (> 85%). Results of the phylogenetic analysis of this COI gene were congruent with the morphological identification and provided strong support for a single clade of local S. medanensis.

Matched MeSH terms: Polymerase Chain Reaction/methods
PCR fingerprinting of Blastocystis isolated from symptomatic and asymptomatic human hosts

Tan TC, Suresh KG, Thong KL, Smith HV

Parasitol Res, 2006 Sep;99(4):459-65.
PMID: 16628457

Genomic DNA from 16 Blastocystis hominis isolates comprising of eight asymptomatic isolates (A1-A8) and eight symptomatic isolates (S1-S8) was amplified by arbitrarily primed polymerase chain reaction (AP-PCR) using 38 arbitrary 10-mer primers. Six primers (A10, B5, C20, D1, F6, and F10) generated reproducible DNA fingerprints. AP-PCR amplification revealed similar DNA fingerprints among all symptomatic isolates (S1-S8) with common bands at 850 bp using primer A10, 920 bp using primer B5, and 1.3 kbp using primer D1. Isolates A1, A3, A4, A5, A6, and A7 showed similar DNA banding patterns and all asymptomatic isolates (A1-A8) shared a major band at 1 kbp using primer B5. Isolates A2 and A8 showed distinct DNA banding patterns that differed from the remainder of the isolates. The results of the phylogenetic analyses showed that all symptomatic isolates (S1-S8) formed a clade with >70% similarity among the isolates and which were clearly separate from asymptomatic isolates A1, A3, A4, A5, A6, and A7. Asymptomatic isolates A2 and A8 formed two distinct and separate clades. AP-PCR revealed higher genetic variability within the asymptomatic isolates than within the symptomatic isolates. The present study suggests that AP-PCR can be a valuable method for differentiating between isolates of B. hominis and our results support the hypothesis that our asymptomatic and symptomatic B. hominis isolates may represent two different strains/species with varying pathogenic potential.

Matched MeSH terms: Polymerase Chain Reaction/methods*
PCR cycle sequencing protocol for population mitochondrial cytochrome b analysis

Lau CH, Yusoff K, Tan SG, Yamada Y

Biotechniques, 1995 Feb;18(2):262-6.
PMID: 7727128

Laboratories intending to adopt cycle sequencing of PCR products in their routine analysis often face a confusing range of methods and kits. Through the study of mitochondrial cytochrome b, we have shown that clean and highly reproducible sequences could be obtained by using a combination of existing simple and economical methods in the preparation of DNA templates, PCR, purification of PCR products and sequencing. Our protocol is useful in itself or as a standard in typing other PCR-amplified DNA at the population level.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Fulltext Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA

Rosli MK, Zamzuriada AS, Syed-Shabthar SM, Mahani MC, Abas-Mazni O, Md-Zain BM

Genet. Mol. Res., 2011;10(4):2554-68.
PMID: 22033937 DOI: 10.4238/2011.October.19.2

PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.

Matched MeSH terms: Polymerase Chain Reaction/methods*
One-step species-specific high resolution melting analysis for nosocomial bacteria detection

Wong YP, Chua KH, Thong KL

J Microbiol Methods, 2014 Dec;107:133-7.
PMID: 25307691

Nosocomial infections are a major public health concern worldwide. Early and accurate identification of nosocomial pathogens which are often multidrug resistant is crucial for prompt treatment. Hence, an alternative real-time polymerase chain reaction coupled with high resolution melting-curve analysis (HRMA) was developed for identification of five nosocomial bacteria. This assay targets species-specific regions of each nosocomial bacteria and produced five distinct melt curves with each representing a particular bacterial species. The melting curves were characterized by peaks of 78.8 ± 0.2 °C for Acinetobacter baumannii, 82.7 ± 0.2 °C for Escherichia coli, 86.3 ± 0.3 °C for Klebsiella pneumoniae, 88.8 ± 0.2 °C for Pseudomonas aeruginosa and 74.6 ± 02 °C for methicillin-resistant Staphylococcus aureus. The assay was able to specifically detect the five bacterial species with an overall detection limit of 2 × 10(-2) ng/μL. In conclusion, the HRM assay developed is a simple and rapid method for identification of the selected nosocomial pathogens.

Matched MeSH terms: Polymerase Chain Reaction/methods*
One-step differential detection of Salmonella enterica serovar Typhi, serovar Paratyphi A and other Salmonella spp. by using a quadruplex real-time PCR assay

Teh CSJ, Lau MY, Chong CW, Ngoi ST, Chua KH, Lee WS, et al.

J Microbiol Methods, 2021 04;183:106184.
PMID: 33662480 DOI: 10.1016/j.mimet.2021.106184

Diseases caused by typhoidal and non-typhoidal Salmonella remain a considerable threat to both developed and developing countries. Based on the clinical symptoms and serological tests, it is sometimes difficult to differentiate the Salmonella enterica serovar Paratyphi A (S. enterica serovar Paratyphi A) from serovar Typhi (S. enterica serovar Typhi). In this study, we developed a quadruplex real-time polymerase chain reaction (PCR) assay with an internal amplification control (IAC), to simultaneously differentiate S. enterica serovar Paratyphi A from serovar Typhi and to detect other Salmonella serovars which cause salmonellosis in humans. This assay was evaluated on 155 salmonellae and non-salmonellae strains and demonstrated 100% specificity in species differentiation. Inclusion of an IAC did not affect the efficiency of the assay. Further evaluation using a blind test on spiked stool, blood and food specimens showed that the detection limit was at 103 -104 CFU/mL (or g) and a high PCR efficiency with different targets (R2 > 0.99), except for S. enterica serovar Paratyphi A in blood. This assay has been applied to clinical specimens to detect the causative agents of gastrointestinal infections and has successfully identified 6 salmonellosis patients from the 50 diarrhoea patients. The quadruplex real-time PCR developed in this study could enhance the detection and differentiation of salmonellae. This assay could be applied to stools, blood and food based on the notable performance in the simulation tests and field evaluation.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
Nucleoprotein gene analysis of fixed and street rabies virus variants using RT-PCR

Arai YT, Yamada K, Kameoka Y, Horimoto T, Yamamoto K, Yabe S, et al.

Arch Virol, 1997;142(9):1787-96.
PMID: 9672637

A simple and rapid single-step reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the nucleoprotein (N) gene of 11 rabies viruses. A conserved set of RT-PCR primers was designed to amplify the most variable region in the N gene. N gene regions were amplified from 6 fixed laboratory viruses, 4 street viruses from dogs in Thailand, and a horse in Zambia. Sequences of the amplified products, together with the database of 91 additional sequences, were analyzed by using PILEUP program of the GCG package. The rabies viruses grouped into at least 9 distinct clusters by < 90% nucleotide similarity of the N gene region: I (4 isolates, USA), II (2 isolates, South America), III (3 isolates, Africa), IV (52 strains, Europe, Middle East, Africa and South America), V (16 isolates, North America and Arctic), VI (17 isolates, Africa), VII (1 isolate, Africa), VIII (6 isolates, Thailand and Malaysia) and IX (1 isolate, Sri Lanka). A unique group of rabies viruses from Thailand and clusters of isolates corresponding to their geographic origin also were determined. The simple and rapid single-step RT-PCR proved to be useful for identifying rabies viruses, and for grouping the viruses into clades by sequence analysis.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Novel multiplex PCR-RFLP assay discriminates bovine, porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells

Sultana S, Hossain MAM, Naquiah NNA, Ali ME

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2018 Sep;35(9):1662-1673.
PMID: 30028648 DOI: 10.1080/19440049.2018.1500719

Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus' consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
Fulltext Non-protein coding RNA genes as the novel diagnostic markers for the discrimination of Salmonella species using PCR

Nithya R, Ahmed SA, Hoe CH, Gopinath SC, Citartan M, Chinni SV, et al.

PLoS One, 2015;10(3):e0118668.
PMID: 25774907 DOI: 10.1371/journal.pone.0118668

Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to humans through contaminated food or water. It is of paramount importance, to generate accurate detection methods for discriminating the various Salmonella species that cause severe infection in humans, including S. Typhi and S. Paratyphi A. Here, we formulated a strategy of detection and differentiation of salmonellosis by a multiplex polymerase chain reaction assay using S. Typhi non-protein coding RNA (sRNA) genes. With the designed sequences that specifically detect sRNA genes from S. Typhi and S. Paratyphi A, a detection limit of up to 10 pg was achieved. Moreover, in a stool-seeding experiment with S. Typhi and S. Paratyphi A, we have attained a respective detection limit of 15 and 1.5 CFU/mL. The designed strategy using sRNA genes shown here is comparatively sensitive and specific, suitable for clinical diagnosis and disease surveillance, and sRNAs represent an excellent molecular target for infectious disease.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
Neuro-genetic optimization of temperature control for a continuous flow polymerase chain reaction microdevice

Lee HW, Arunasalam P, Laratta WP, Seetharamu KN, Azid IA

J Biomech Eng, 2007 Aug;129(4):540-7.
PMID: 17655475

In this study, a hybridized neuro-genetic optimization methodology realized by embedding finite element analysis (FEA) trained artificial neural networks (ANN) into genetic algorithms (GA), is used to optimize temperature control in a ceramic based continuous flow polymerase chain reaction (CPCR) device. The CPCR device requires three thermally isolated reaction zones of 94 degrees C, 65 degrees C, and 72 degrees C for the denaturing, annealing, and extension processes, respectively, to complete a cycle of polymerase chain reaction. The most important aspect of temperature control in the CPCR is to maintain temperature distribution at each reaction zone with a precision of +/-1 degree C or better, irrespective of changing ambient conditions. Results obtained from the FEA simulation shows good comparison with published experimental work for the temperature control in each reaction zone of the microfluidic channels. The simulation data are then used to train the ANN to predict the temperature distribution of the microfluidic channel for various heater input power and fluid flow rate. Once trained, the ANN analysis is able to predict the temperature distribution in the microchannel in less than 20 min, whereas the FEA simulation takes approximately 7 h to do so. The final optimization of temperature control in the CPCR device is achieved by embedding the trained ANN results as a fitness function into GA. Finally, the GA optimized results are used to build a new FEA model for numerical simulation analysis. The simulation results for the neuro-genetic optimized CPCR model and the initial CPCR model are then compared. The neuro-genetic optimized model shows a significant improvement from the initial model, establishing the optimization method's superiority.

Matched MeSH terms: Polymerase Chain Reaction/methods*

Filters

Please provide feedback to Administrator (afdal@afpm.org.my)

External Links