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Fulltext Development of TaqMan-based real-time RT-PCR assay based on N gene for the quantitative detection of feline morbillivirus

Makhtar ST, Tan SW, Nasruddin NA, Abdul Aziz NA, Omar AR, Mustaffa-Kamal F

BMC Vet Res, 2021 Mar 23;17(1):128.
PMID: 33757494 DOI: 10.1186/s12917-021-02837-6

BACKGROUND: Morbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.
RESULTS: A specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34-0.53% and 1.38-2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.
CONCLUSIONS: The TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.

Matched MeSH terms: Real-Time 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
Identification of male-specific SNP markers and development of PCR-based genetic sex identification technique in crucifix crab (Charybdis feriatus) with implication of an XX/XY sex determination system

Fang S, Zhang Y, Shi X, Zheng H, Li S, Zhang Y, et al.

Genomics, 2020 01;112(1):404-411.
PMID: 30851358 DOI: 10.1016/j.ygeno.2019.03.003

In this study, we first identified male-specific SNP markers using restriction site-associated DNA sequencing, and further developed a PCR-based sex identification technique for Charybdis feriatus. A total of 296.96 million clean reads were obtained, with 114.95 and 182.01 million from females and males. After assembly and alignment, 10 SNP markers were identified being heterozygous in males but homozygous in females. Five markers were further confirmed to be male-specific in a large number of individuals. Moreover, two male-specific sense primers and a common antisense primer were designed, using which, a PCR-based genetic sex identification method was successfully developed and used to identify the sex of 103 individuals, with a result of 49 females and 54 males. The presence of male-specific SNP markers suggests an XX/XY sex determination system for C. feriatus. These findings should be helpful for better understanding sex determination mechanism, and drafting artificial breeding program in crustaceans.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Fulltext Screening of 22q11.2DS Using Multiplex Ligation-Dependent Probe Amplification as an Alternative Diagnostic Method

Maran S, Faten SA, Lim SE, Lai KS, Ibrahim WPW, Ankathil R, et al.

Biomed Res Int, 2020;2020:6945730.
PMID: 33062692 DOI: 10.1155/2020/6945730

Background: The 22q11.2 deletion syndrome (22q11.2DS) is the most common form of deletion disorder in humans. Low copy repeats flanking the 22q11.2 region confers a substrate for nonallelic homologous recombination (NAHR) events leading to rearrangements which have been reported to be associated with highly variable and expansive phenotypes. The 22q11.2DS is reported as the most common genetic cause of congenital heart defects (CHDs).
Methods: A total of 42 patients with congenital heart defects, as confirmed by echocardiography, were recruited. Genetic molecular analysis using a fluorescence in situ hybridization (FISH) technique was conducted as part of routine 22q11.2DS screening, followed by multiplex ligation-dependent probe amplification (MLPA), which serves as a confirmatory test.
Results: Two of the 42 CHD cases (4.76%) indicated the presence of 22q11.2DS, and interestingly, both cases have conotruncal heart defects. In terms of concordance of techniques used, MLPA is superior since it can detect deletions within the 22q11.2 locus and outside of the typically deleted region (TDR) as well as duplications.
Conclusion: The incidence of 22q11.2DS among patients with CHD in the east coast of Malaysia is 0.047. MLPA is a scalable and affordable alternative molecular diagnostic method in the screening of 22q11.2DS and can be routinely applied for the diagnosis of deletion syndromes.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
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: Polymerase Chain Reaction/methods*
A simple 18S rDNA approach for the identification of cultured eukaryotic microalgae with an emphasis on primers

Khaw YS, Khong NMH, Shaharuddin NA, Yusoff FM

J Microbiol Methods, 2020 05;172:105890.
PMID: 32179080 DOI: 10.1016/j.mimet.2020.105890

Any forms of valorization of microorganisms would require accurate identity recognition to ensure repeatability, reproducibility and quality assurance. This study aimed to evaluate the effectiveness of different primers for identifying cultured eukaryotic microalgae using a simple 18S rDNA approach. A total of 34 isolated microalgae and one culture collection were utilized in the search for an effective molecular identification method for microalgae. Ammonium formate was applied to marine microalgae prior to DNA extraction. The microalgal DNA was extracted using a commercial kit and subjected directly to PCR amplification using four different published 18S rDNA primers. The DNA sequences were analysed using Basic Local Alignment Search Tool (BLAST) and phylogenetic trees to determine the microalgae identity. The identity was further validated with conventional morphological taxonomic identification, and the relationship of microalgal morphology and genetic materials was also determined. The microalgal DNA was successfully amplified, including marine species without prior cleaning. In addition, the ss5 + ss3 primer pair was found to be an ideal primer set among the tested primers for identifying microalgae. Overall, molecular identification showed relative matching with morphological identification (82.86%). This study is important because it serves as a platform to develop a standardized eukaryotic microalgae identification method. In addition, this method could help to ease the eukaryotic microalgae identification process and enrich the current reference databases such as GenBank.

Matched MeSH terms: Polymerase Chain Reaction/methods
Human T-cell receptor V gene segment of alpha and beta families: A revised primer design strategy

Ch'ng ACW, Chan SK, Ignatius J, Lim TS

Eur J Immunol, 2019 08;49(8):1186-1199.
PMID: 30919413 DOI: 10.1002/eji.201747328

The application of human TCR in cancer immunotherapy has gained momentum with developments in tumor killing strategies using endogenous adaptive immune responses. The successful coverage of a diverse TCR repertoire is mainly attributed to the primer design of the human TCR V genes. Here, we present a refined primer design strategy of the human TCR V gene by clustering V gene sequence homolog for degenerate primer design based on the data from IMGT. The primers designed were analyzed and the PCR efficiency of each primer set was optimized. A total of 112 alpha and 160 beta sequences were aligned and clustered using a phylogram yielding 32 and 27 V gene primers for the alpha and beta family. The new primer set was able to provide 93.75% and 95.63% coverage for the alpha and beta family, respectively. A semi-qualitative approach using the designed primer set was able to provide a relative view of the TCR V gene diversity in different populations. Taken together, the new primers provide a more comprehensive coverage of the TCR gene diversity for improved TCR library generation and TCR V gene analysis studies.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Fulltext Evaluation of PCR-based approach for serotype determination of Streptococcus pneumoniae

Shakrin NN, Balasubramaniam SD, Yusof HA, Mastuki MF, Masri SN, Taib NM, et al.

Trop Biomed, 2013 Jun;30(2):338-44.
PMID: 23959499 MyJurnal

Determination of Streptococcus pneumoniae serotypes is essential for epidemiological surveillance. Therefore accurate, reliable and cost effective serotyping method is crucial. In this study, we determined the serotypes of 41 pneumococcal isolates recovered from human anterior nares by multiplex Polymerase Chain Reaction (PCR) utilizing published primers. The data was then compared with conventional serology using latex agglutination (LA) and the Quellung reaction. Based on the PCR-approach, 8 different serogroups/serotypes were detected with one isolate classified as non-typeable (cpsA-negative). In reference to the serology-based data, the results were in agreement except for one isolate. For the latter isolate, the LA and Quellung tests failed to show a reaction but the PCR-approach and sequencing identified the isolate as serogroup 15B/C. Based on this experimental setting, we found that the PCR-approach for pneumococcal serotypes determination is reliable to serve as the alternative for determining the pneumococcal serotyping.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
Fulltext Determination of toxinotypes of environmental Clostridium perfringens by Polymerase Chain Reaction

Florence L CH, Hakim SL, Kamaluddin MA, Thong KL

Trop Biomed, 2011 Apr;28(1):171-4.
PMID: 21602783

Toxinotype of Clostridium perfringens (CP) isolates collected from the Bernam River, Selangor River and Tengi Canal between April 2007 and January 2008 were determined by Polymerase Chain Reaction (PCR) using published primers. All the 147 isolates were toxinotype Type A, harbouring the alpha toxin gene. In addition, 5 of the isolates also had the enterotoxin (CPE) gene.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Fulltext Comparison of conventional versus real-time PCR detection of Brugia malayi DNA from dried blood spots from school children in a low endemic area

Rahmah N, Nurulhasanah O, Norhayati S, Zulkarnain I, Norizan M

Trop Biomed, 2010 Apr;27(1):54-9.
PMID: 20562814 MyJurnal

Microscopic detection of active phase of lymphatic filariasis is indicated by the presence of microfilaria in whole blood. This method is not sensitive and requires relatively large amount of blood sample. PCR allows very sensitive detection of the parasite DNA using a smaller amount of blood; and the use of dried blood spots facilitates sample transportation. Nevertheless, limited studies have been reported on PCR using dried blood spot for detection of Brugia malayi. In this study, we investigated the effects of concentrating whole blood genomic DNA sample and the amplification methods [conventional PCR (C-PCR) and real-time PCR] on the detection of B. malayi DNA from dried blood spots from a very low endemic area in Malaysia. Both C-PCR and real-time PCR detected 2 out of 18 (11%) samples as positive from non-concentrated genomic DNA preparations. After the DNA samples were pooled and concentrated, both C-PCR and realtime PCR detected B. malayi DNA amplifications in 7 out of 18 (39%) samples. However one sample which showed faint band in C-PCR was detected as highly positive in real-time PCR. In conclusion, both C-PCR and real-time PCR using dried blood spots from a low endemic area demonstrated equal sensitivity for detection of B. malayi DNA.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Fulltext Use of molecular tools to distinguish Entamoeba histolytica and Entamoeba dispar infection among the aborigines in Cameron Highlands

Noor Azian MY, Lokman Hakim S, Maslawaty MN

Trop Biomed, 2006 Jun;23(1):31-6.
PMID: 17041549 MyJurnal

Amoebiasis is an infectious diseased caused by parasitic one-celled protozoan called Entamoeba histolytica. Numerous protozoa also can inhabit the gastro-intestinal tract of human. Majority of these protozoa are non-pathogenic commensals or only causes disease under certain circumstances. Morphologically, E. histolytica, the invasive form, share the same characteristic with the nonpathogenic form, E. dispar. Both strains can be distinguished by using DNA identification. Many previous researches in Malaysia only reported infection with E. histolytica infection. Therefore in this study we tried to classify infection among the aborigines in Cameron Highland as true E. histolytica or E. dispar by Nested Polymerase Chain Reaction (Nested PCR) and Restriction enzyme (RE) digestion. Results showed that 31 samples were positive by microscopic examination, however of these 28 (13.2%) samples were positive for E. histolytica and 12 (5.6%) samples were positive for E. dispar by molecular tools.

Matched MeSH terms: Polymerase Chain Reaction/methods
Fulltext Rapid detection of chikungunya virus in laboratory infected Aedes aegypti by Reverse-Transcriptase- Polymerase Chain Reaction (RT-PCR)

Rohani A, Yulfi H, Zamree I, Lee HL

Trop Biomed, 2005 Dec;22(2):149-54.
PMID: 16883281 MyJurnal

A study of chikungunya virus was carried out to establish Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR) as a rapid detection technique of the virus. The susceptibility of lab-colonized Aedes aegypti to chikungunya virus was also determined. Artificial membrane feeding technique was used to orally feed the mosquitoes with a human isolate of chikungunya virus. A total of 100 fully engorged female Ae. aegypti were obtained and maintained for 7 days. Seventy of them survived and then pooled at 10 individuals per pool. Total RNA was extracted from the samples and RT-PCR amplifications were carried out. Five out of 7 pools showed positive PCR band at 350-bp, indicating Ae. aegypti is a potential vector of chikungunya virus. The minimum infection rate (MIR) was 71% within these laboratory colonies. RT-PCR is a sensitive technique that is useful in detecting infected mosquitoes in epidemic areas. This technique can de used as a rapid detection method and provide an early virologic surveillance systems of chikungunya virus infected mosquitoes.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Rapid detection of α-thalassaemia variants using droplet digital PCR

Lee TY, Lai MI, Ramachandran V, Tan JA, Teh LK, Othman R, et al.

Int J Lab Hematol, 2016 Aug;38(4):435-43.
PMID: 27349818 DOI: 10.1111/ijlh.12520

INTRODUCTION: Alpha thalassaemia is a highly prevalent disease globally and is a well-known public health problem in Malaysia. The deletional forms of the mutation are the most common forms found in alpha thalassaemia. The three most common deletional alpha thalassaemia found in this region include --(SEA) deletion, -α(3.7) rightward and -α(4.2) leftward deletions. The prevalence rate of triplication alpha cases such as ααα(anti3.7) and ααα(anti4.2) is not known in Malaysia although it plays a role in exacerbating the clinical phenotypes in beta thalassaemia carriers. Recently, there have been more reported cases of rare alpha thalassaemia mutations due to the advancement of molecular techniques involved in thalassaemia detections. Therefore, it is essential to develop a new method which allows the detection of different alpha thalassaemia mutations including the rare ones simultaneously and accurately.
METHODS: The purpose of this study was to design an assay for the detection of triplications, common and rare deletional alpha thalassaemia using droplet digital PCR (ddPCR).
RESULTS: This is a quantitative detection method to measure the changes of copy number which can detect deletions, duplications and triplications of the alpha globin gene simultaneously.
CONCLUSION: In conclusion, ddPCR is an alternative method for rapid detection of alpha thalassaemia variants in Malaysia.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Fulltext Molecular Detection of Methicillin-Resistant Staphylococcus aureus by Non-Protein Coding RNA-Mediated Monoplex Polymerase Chain Reaction

Soo Yean CY, Selva Raju K, Xavier R, Subramaniam S, Gopinath SC, Chinni SV

PLoS One, 2016;11(7):e0158736.
PMID: 27367909 DOI: 10.1371/journal.pone.0158736

Non-protein coding RNA (npcRNA) is a functional RNA molecule that is not translated into a protein. Bacterial npcRNAs are structurally diversified molecules, typically 50-200 nucleotides in length. They play a crucial physiological role in cellular networking, including stress responses, replication and bacterial virulence. In this study, by using an identified npcRNA gene (Sau-02) in Methicillin-resistant Staphylococcus aureus (MRSA), we identified the Gram-positive bacteria S. aureus. A Sau-02-mediated monoplex Polymerase Chain Reaction (PCR) assay was designed that displayed high sensitivity and specificity. Fourteen different bacteria and 18 S. aureus strains were tested, and the results showed that the Sau-02 gene is specific to S. aureus. The detection limit was tested against genomic DNA from MRSA and was found to be ~10 genome copies. Further, the detection was extended to whole-cell MRSA detection, and we reached the detection limit with two bacteria. The monoplex PCR assay demonstrated in this study is a novel detection method that can replicate other npcRNA-mediated detection assays.

Matched MeSH terms: Polymerase Chain Reaction/methods*
High-Throughput RT-qPCR for the Analysis of Circulating MicroRNAs

Tan GW, Tan LP

Methods Mol Biol, 2017;1580:7-19.
PMID: 28439823 DOI: 10.1007/978-1-4939-6866-4_2

Reverse transcription followed by real-time or quantitative polymerase chain reaction (RT-qPCR) is the gold standard for validation of results from transcriptomic profiling studies such as microarray and RNA sequencing. The current need for most studies, especially biomarker studies, is to evaluate the expression levels or fold changes of many transcripts in a large number of samples. With conventional low to medium throughput qPCR platforms, many qPCR plates would have to be run and a significant amount of RNA input per sample will be required to complete the experiments. This is particularly challenging when the size of study material (small biopsy, laser capture microdissected cells, biofluid, etc.), time, and resources are limited. A sensitive and high-throughput qPCR platform is therefore optimal for the evaluation of many transcripts in a large number of samples because the time needed to complete the entire experiment is shortened and the usage of lab consumables as well as RNA input per sample are low. Here, the methods of high-throughput RT-qPCR for the analysis of circulating microRNAs are described. Two distinctive qPCR chemistries (probe-based and intercalating dye-based) can be applied using the methods described here.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain

Hossain MAM, Ali ME, Sultana S, Asing, Bonny SQ, Kader MA, et al.

J Agric Food Chem, 2017 May 17;65(19):3975-3985.
PMID: 28481513 DOI: 10.1021/acs.jafc.7b00730

Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

Matched MeSH terms: Real-Time 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*
Multiplex real-time RT-PCR assay for transfusion transmitted viruses in sero-negative allogeneic blood donors: an experience from Southern Pakistan

Sultan S, Nasir MI, Rafiq S, Baig MA, Akbani S, Irfan SM

Malays J Pathol, 2017 Aug;39(2):149-154.
PMID: 28866696

BACKGROUND: Blood transfusion safety commences with healthy donor recruitment. The threat of transfusion transmitted infections is greatly minimized by serological tools but not entirely eliminated. Recently, nucleic-acid testing for blood donor screening has virtually eliminated this jeopardy.
METHODS: This prospective study was conducted from February 2015 to February 2016. Samples from seronegative donors were run on multiplex assay (Cobas, S-201 system platform, Roche) in a batch of six [MP-NAT]. In case of reactive pool, tests were run on every individual sample [IDNAT].
RESULTS: Of 16957 donors, 16836 (99.2%) were replacement donors and the remaining 121 (0.7%) were voluntary donors, with a mean age of 29.09 ± 7.04 years. After serologic screening of all 16957 donors, 955 (5.6%) were found to be reactive; 291(1.71%) were reactive for hepatitis-B surface antigen, 361 (2.12%) for antibody to hepatitis C virus (anti-HCV), 14 (0.08%) for antibody to human immunodeficiency virus, 287 (1.69%) for syphilis and 2 (0.01%) for malaria. 14 (0.08%) NAT reactive donors were identified after testing the 16002 seronegative donors, with an overall NAT yield of one reactivity out of 1143 blood donations; 10 donors for HBV-DNA (HBV NAT yield-1:1600) and remaining 4 for HCV-RNA (HCV-NAT yield-1:4000). None were HIV positive.
CONCLUSION: NAT has improved the safety attributes in blood products. Although the positivity rate for NAT testing is low but in view of the high prevalence of transfusion transmitted infections in our country, we recommend the parallel use of both serology and NAT screening of all donated blood.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
Analysis of four PCR/SNaPshot multiplex assays analyzing 52 SNPforID markers

Goodwin W, Alimat S

Electrophoresis, 2017 04;38(7):1007-1015.
PMID: 28008628 DOI: 10.1002/elps.201600383

The SNPforID consortium identified a panel of 52 SNPs for forensic analysis that has been used by several laboratories worldwide. The original analysis of the 52 SNPs was based on a single multiplex reaction followed by two single-base-extension (SBE) reactions each of which was analyzed using capillary electrophoresis. The SBE assays were designed for high throughput genetic analyzers and were difficult to use on the single capillary ABI PRISM 310 Genetic Analyzer and the latest generation 3500 Genetic Analyzer, as sensitivity on the 310 was low and separation of products on the 3500 with POP-7™ was poor. We have modified the original assay and split it into four multiplex reactions, each followed by an SBE assay. These multiplex assays were analyzed using polymer POP-4™ on ABI 310 PRISM® and polymers POP-4™, POP-6™ and POP-7™ on the 3500 Genetic Analyzer. The assays were sensitive and reproducible with input DNA as low as 60 pg using both the ABI 310 and 3500. In addition, we found that POP-6™ was most effective with the 3500, based on the parameters that we assessed, achieving better separation of the small SBE products; this conflicted with the recommended use of POP-7™ by the instrument manufacturer. To support the use of the SNP panel in casework in Malaysia we have created an allele frequency database from 325 individuals, representing the major population groups within Malaysia. Population and forensic parameters were estimated for all populations and its efficacy evaluated using 51 forensic samples from challenging casework.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*
Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

Hussain H, Chong NF

Biomed Res Int, 2016;2016:8041532.
PMID: 27995143

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.

Matched MeSH terms: Multiplex Polymerase Chain Reaction/methods*

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