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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*
Rapid concentration and detection of hepatitis A virus from lettuce and strawberries

Bidawid S, Farber JM, Sattar SA

J Virol Methods, 2000 Aug;88(2):175-85.
PMID: 10960705

Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 microl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the IM-PCR. However, considering the large volumes (< or =50 ml) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to < or =20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Fulltext Validation of a multiplex RT-PCR assay for screening significant oncogene fusion transcripts in children with acute lymphoblastic leukaemia

Ariffin H, Chen SP, Wong HL, Yeoh A

Singapore Med J, 2003 Oct;44(10):517-20.
PMID: 15024455

In childhood acute lymphoblastic leukaemia (ALL), cytogenetics play an important role in diagnosis, allocation of treatment and prognosis. Conventional cytogenetic analysis, involving mainly karyotyping in our experience, has not been successful in a large proportion of cases due to inadequate metaphase spreads and poor chromosome morphology. Our aim is to develop a highly sensitive and specific method to screen simultaneously for the four most frequent fusion transcripts resulting from specific chromosomal translocations, namely, both the CML- and ALLtype BCR-ABL transcripts of t(9;22), E2A-PBX1 transcript of t(1;19), the MLL-AF4 transcript of t(4;11) and TEL-AML1 (also termed ETV6-CBFA2) of the cryptic t(12;21). A multiplex reverse transcription polymerase chain reaction protocol (RT-PCR) was developed and tested out on archival bone marrow samples and leukaemia cell lines. In all samples with a known translocation detected by cytogenetic techniques, the same translocation was identified by the multiplex-PCR assay. Multiplex RT-PCR assay is an effective, sensitive, accurate and cost-effective diagnostic tool which can improve our ability to accurately and rapidly risk-stratify patients with childhood ALL.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
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*
Incorrect identification of recent Asian strains of Coxsackievirus A16 as human enterovirus 71: improved primers for the specific detection of human enterovirus 71 by RT PCR

Perera D, Podin Y, Akin W, Tan CS, Cardosa MJ

BMC Infect Dis, 2004 May 4;4:11.
PMID: 15122971

Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Implementation in Australia of molecular diagnostic techniques for the rapid detection of foot and mouth disease virus

Boyle DB, Taylor T, Cardoso M

Aust. Vet. J., 2004 Jul;82(7):421-5.
PMID: 15354851

OBJECTIVE: To evaluate and implement rapid molecular diagnostic techniques for the detection of foot and mouth disease virus (FMDV) suitable for use in Australia.
DESIGN: Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks.
RESULTS: Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3.
CONCLUSION: Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Specific detection of Nipah virus using real-time RT-PCR (TaqMan)

Guillaume V, Lefeuvre A, Faure C, Marianneau P, Buckland R, Lam SK, et al.

J Virol Methods, 2004 Sep 15;120(2):229-37.
PMID: 15288966

Nipah and Hendra viruses belong to the novel Henipavirus genus of the Paramyxoviridae family. Its zoonotic circulation in bats and recent emergence in Malaysia with fatal consequences for humans that were in close contact with infected pigs, has made the reinforcement of epidemiological and clinical surveillance systems a priority. In this study, TaqMan RT-PCR of the Nipah nucleoprotein has been developed so that Nipah virus RNA in field specimens or laboratory material can be characterized rapidly and specifically and quantitated. The linearity of the standard curve allowed quantification of 10(3) to 10(9) RNA transcripts. The sensitivity of the test was close to 1 pfu. The kinetics of Nipah virus production in Vero cells was monitored by the determination of infectious virus particles in the supernatant fluid and by quantitation of the viral RNA. Approximately, 1000 RNA molecules were detected per virion, suggesting the presence of many non-infectious particles, similar to other RNA viruses. TaqMan real-time RT-PCR failed to detect Hendra virus DNA. Importantly, the method was able to detect virus despite a similar ratio in viremic sera from hamsters infected with Nipah virus. This standardized technique is sensitive and reliable and allows rapid detection and quantitation of Nipah RNA in both field and experimental materials used for the surveillance and specific diagnosis of Nipah virus.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
A simple multiplex PCR method for the concurrent detection of three CYP2C8 variants

Muthiah YD, Lee WL, Teh LK, Ong CE, Salleh MZ, Ismail R

Clin Chim Acta, 2004 Nov;349(1-2):191-8.
PMID: 15469873 DOI: 10.1016/j.cccn.2004.06.024

BACKGROUND: Cytochrome P450 (CYP) 2C8 is a principle enzyme responsible for the metabolism of many clinically important drugs as well as endogenous compounds such as arachidonic acid. The enzyme is genetically polymorphic but a simple method is not available to study its genetic polymorphism. We developed and optimized a variant-specific PCR techniques to detect CYP2C8*2, CYP2C8*3 and CYP2C8*4.
METHOD: Genomic DNA was extracted from blood using standard extraction methods. A two-step PCR method was developed to detect simultaneously three CYP2C8 variants. In the first PCR (PCR1), specific regions from exons 3, 5 and 8 of the CYP2C8 gene were amplified. The products were used as templates in parallel alleles-specific PCR (PCR2). This method was tested against DNA samples obtained from 57 healthy Malaysian volunteers.
RESULT: The bands of interest were successfully amplified. This method showed specific and reproducible results when tested on healthy volunteers. DNA sequencing further confirmed genotype results obtained from current method.
CONCLUSION: We have successfully developed and optimized a multiplex PCR method suitable for use in population studies of CYP2C8 polymorphism.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4

Sunderasan E, Bahari A, Arif SA, Zainal Z, Hamilton RG, Yeang HY

Clin Exp Allergy, 2005 Nov;35(11):1490-5.
PMID: 16297147 DOI: 10.1111/j.1365-2222.2005.02371.x

BACKGROUND:
Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information.

OBJECTIVE:
We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding.

METHODS:
The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients.

RESULTS:
The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue.

CONCLUSION:
The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA.

Matched MeSH terms: Reverse Transcriptase 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*
Male-specific RNA coliphages detected by plaque assay and RT-PCR in tropical river waters and animal fecal matter

Yee SY, Fong NY, Fong GT, Tak OJ, Hui GT, Su Ming Y

Int J Environ Health Res, 2006 Feb;16(1):59-68.
PMID: 16507481

Male-specific RNA coliphages (FRNA) have been recommended as indicators of fecal contamination and of the virological quality of water. In this study, 16 river water and 183 animal fecal samples were examined for the presence of FRNA coliphages by a plaque assay using Salmonella typhimurium WG49 and WG25 to differentiate between male-specific and somatic phages, a RNase spot test to differentiate between DNA and RNA phages and a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific identification of FRNA phages. The overall recovery rate for F-specific coliphages was 8.0%. (4.4% from animal fecal matter and 50% from river water samples). Plaque counts were generally low (< 6 x 10(2) pfu per g feces or ml water), with FRNA (6.5%) and Male-specific DNA coliphages (FDNA) (7.0%) phages occurring at almost equal frequencies. The RT-PCR was positive in all FRNA plaques and was able to identify FRNA phages in mixed populations of FRNA, FDNA and somatic phages.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Fulltext Isolation of monoclonal antibodies-escape variant of dengue virus serotype 1

Chua SK, Selvanesan S, Sivalingam B, Chem YK, Norizah I, Zuridah H, et al.

Singapore Med J, 2006 Nov;47(11):940-6.
PMID: 17075660

During an outbreak from December 2004 to March 2005, 138 isolates of dengue virus were prospectively obtained from acute-phase serum samples of 1,067 patients with the provisional clinical diagnosis of acute dengue illness admitted to the adult wards of Hospital Tengku Ampuan Rahimah, Klang, Malaysia. Of the 138 dengue virus isolates, 87, 11, 24 and 3 were typed as dengue serotypes 1, 2, 3 and 4, respectively, by a commercial dengue virus typing kit using monoclonal antibodies (Mab). 13 dengue virus isolates could not be assigned to any specific serotype by serotyping Mab and molecular typing using dengue-type specific molecular typing primer pairs. We report the associated clinical features and limited molecular genetics of this Mab-escape dengue virus variant.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR

Kong YY, Thay CH, Tin TC, Devi S

J Virol Methods, 2006 Dec;138(1-2):123-30.
PMID: 17000012 DOI: 10.1016/j.jviromet.2006.08.003

The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay

Ng LF, Barr I, Nguyen T, Noor SM, Tan RS, Agathe LV, et al.

BMC Infect Dis, 2006;6:40.
PMID: 16512903

Continuous outbreaks of the highly pathogenic H5N1 avian influenza A in Asia has resulted in an urgent effort to improve current diagnostics to aid containment of the virus and lower the threat of a influenza pandemic. We report here the development of a PCR-based assay that is highly specific for the H5N1 avian influenza A virus.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
A preliminary study of dengue infection in Brunei

Osman O, Fong MY, Devi S

Jpn J Infect Dis, 2007 Jul;60(4):205-8.
PMID: 17642533

The purpose of this study was to examine the extent of dengue infection in Brunei and to determine the predominant serotype circulating in the country. The study generated useful epidemiological data on dengue infection in Brunei. A total of 271 samples from patients suspected of having dengue infections were selected and analyzed. All patients were seen in clinics and hospitals in Brunei. The samples were collected from April 2005 to April 2006 and transported to the WHO Collaborating Centre for Arbovirus Reference and Research, University of Malaya, Malaysia. The following tests were used to achieve the objectives: in-house IgM-capture enzyme-linked immunosorbent assay, virus isolation in mosquito albopictus cell line (C6/36), and viral RNA detection and serotyping by reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that 45 people were positive for dengue-specific IgM (27 males and 18 females), while RT-PCR detected dengue viral RNA in 12 patients, 3 identified as DEN-1 and 9 as DEN-2. Dengue virus was isolated from 6 patients using the C6/36 cell line; 3 were DEN-2 isolates and 3 were DEN-1 isolates. These data show that dengue virus is circulating in Brunei and the predominant infecting serotype for that period was DEN-2 followed by DEN-1. This study is the first to report the detection and isolation of dengue virus from Brunei using RT-PCR and culture in the C6/36 albopictus mosquito cell line.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Fulltext Rapid detection and serotyping of dengue virus by multiplex RT-PCR and real-time SYBR green RT-PCR

Yong YK, Thayan R, Chong HT, Tan CT, Sekaran SD

Singapore Med J, 2007 Jul;48(7):662-8.
PMID: 17609830

Dengue fever and dengue haemorrhagic fever currently rank highly among the newly-emerging infectious diseases, and are considered to be the most important arboviral disease worldwide. The definitive diagnosis is culture analysis, but practical considerations limit its use. Also, the period for viral detection is limited. Within a day or two after fever subsides, rising levels of antibodies interfere with viral cultures. An alternative to this quandary is the use of viral RNA detection assays. In our laboratory, a reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed using a set of degenerate primers.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A

Ong WT, Omar AR, Ideris A, Hassan SS

J Virol Methods, 2007 Sep;144(1-2):57-64.
PMID: 17512062

Avian influenza viruses are pathogens of economical and public health concerns. However, infections caused by low pathogenic avian influenza particularly H9N2 subtype are not associated with clear clinical features. Hence, rapid detection and subtyping of the virus will enable immediate measures to be implemented for preventing widespread transmission. This study highlights the development of a multiplex real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) assay using SYBR Green 1 chemistry for universal detection of avian influenza viruses and specific subtyping of H9N2 isolates based on melting temperatures (T(m)) discriminations. Three melting peaks generated simultaneously at temperatures 85.2+/-1.0, 81.9+/-0.9 and 78.7+/-0.9 degrees C represent NP, H9 and N2 gene products, respectively. The RRT-PCR assay was about 10-100-fold more sensitive when compared to the conventional RT-PCR method using reference H9N2 isolate. In addition, the RRT-PCR assay was 100% sensitive as well as 92% specific according to the standard virus isolation method in detecting experimentally infected specific-pathogen-free (SPF) chickens.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Fulltext Epidemiologic and virologic investigation of hand, foot, and mouth disease, southern Vietnam, 2005

Van Tu P, Thao NTT, Perera D, Truong KH, Tien NTK, Thuong TC, et al.

Emerg Infect Dis, 2007 Nov;13(11):1733-41.
PMID: 18217559 DOI: 10.3201/eid1311.070632

During 2005, 764 children were brought to a large children's hospital in Ho Chi Minh City, Vietnam, with a diagnosis of hand, foot, and mouth disease. All enrolled children had specimens (vesicle fluid, stool, throat swab) collected for enterovirus isolation by cell culture. An enterovirus was isolated from 411 (53.8%) of the specimens: 173 (42.1%) isolates were identified as human enterovirus 71 (HEV71) and 214 (52.1%) as coxsackievirus A16. Of the identified HEV71 infections, 51 (29.5%) were complicated by acute neurologic disease and 3 (1.7%) were fatal. HEV71 was isolated throughout the year, with a period of higher prevalence in October-November. Phylogenetic analysis of 23 HEV71 isolates showed that during the first half of 2005, viruses belonging to 3 subgenogroups, C1, C4, and a previously undescribed subgenogroup, C5, cocirculated in southern Vietnam. In the second half of the year, viruses belonging to subgenogroup C5 predominated during a period of higher HEV71 activity.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods
Rapid detection of the UGT1A1 single nucleotide polymorphism G211A using real-time PCR with Taqman minor groove binder probes

Wong FL, Wang MK, Boo NY, Hamidah NH, Ainoon BO

J Clin Lab Anal, 2007;21(3):167-72.
PMID: 17506482

The UGT1A1 Taqman MGB probe single nucleotide polymorphism (SNP) genotyping assay was developed to detect nucleotide 211 of the UDP-glucoronocyltransferase 1A1 (UGT1A1) gene. Defects in this enzyme interfere with process of conjugation of bilirubin and cause unconjugated hyperbilirubinemia. Variation at nucleotide 211 in the coding region of the UGT1A1 gene has been shown to be prevalent in Japanese and Chinese. Using an ABI sequence detection system (SDS) 7000, an allele-specific real-time PCR-based genotyping method was established to detect nucleotide G211A. Cord blood from 125 infants without hyperbilirubinemia (controls) were compared with cord blood from 74 infants (cases) with severe hyperbilirubinemia (total serum bilirubin > 300 micromol/L). Homozygous variation of the UGT1A1 gene at nucleotide 211(A/A) is significantly more common in cases (14.9%) than in controls (0.8%) (P<0.001). Direct sequencing from 20 randomly selected samples showed eight samples with homozygous wild type, seven with homozygous variant, and five samples were heterozygous. The result from this assay was in complete concordance with the DNA sequencing result and clearly discriminate wild-type (G/G), homozygous variant (A/A), and heterozygous (G/A). This assay is rapid and robust for screening of SNP G211A to determine if this polymorphism plays a role in causing severe neonatal jaundice in the local context.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*
Quantitative RT PCR approach to evaluate the neurogenic and gliagenic gene expression of cultured human amniotic epithelial cells

Tan GC, Simat SF, Abdul Rahman H, Tan AE, Chua KH

Med J Malaysia, 2008 Jul;63 Suppl A:51-2.
PMID: 19024979

The aim of the study is to determine the neuronal and glial gene expression of cultured human amniotic epithelial cells (HAECs) in serial passages. HAECs obtained from human term placentae were cultured in F12:DMEM (1:1) + 10% FBS +10ng/ml EGF in serial passages. Quantitative RT-PCR was used to assess the gene expression analysis. The results showed that the cultured HAECs expressed the neural stem cell genes (Nestin, NSE and Vimentin), mature neuronal genes (TH, MAP-2, beta-III-tubulin and NFM) and glial genes (CNPase, MBP and Olig). These neural stem cell genes increased with serial passages while the genes expression for mature neuronal and glial cells were downregulated. These results suggested that HAECs may promote or involve in neurogenesis and gliagenesis.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/methods*

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