A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals

2009 ◽  
Vol 106 (2) ◽  
pp. 357-365 ◽  
Author(s):  
David O. Odongo ◽  
Jack D. Sunter ◽  
Henry K. Kiara ◽  
Robert A. Skilton ◽  
Richard P. Bishop
Keyword(s):  
Nested Pcr ◽  
Theileria Parva ◽  
Bovine Blood ◽  
Pcr Assay ◽  
Blood Samples ◽  
Enhanced Sensitivity

Detection ofNeospora caninumin aborted bovine fetuses and dam blood samples by nested PCR and ELISA and seroprevalence in Beijing and Tianjin, China

Parasitology ◽  
2009 ◽  
Vol 136 (11) ◽  
pp. 1251-1256 ◽  
Author(s):  
L. YAO ◽  
N. YANG ◽  
Q. LIU ◽  
M. WANG ◽  
W. ZHANG ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Diagnostic Tool ◽  
Nested Pcr ◽  
Neospora Caninum ◽  
Primary Diagnosis ◽  
Dairy Herds ◽  
Pcr Assay ◽  
Blood Samples ◽  
Bovine Fetuses ◽  
The Brain

SUMMARYNeospora caninuminfection is a significant cause of abortion in cattle. We investigated the tissue distribution ofN. caninumin aborted bovine fetuses and dam blood samples by a nested PCR assay, and compared the nested PCR with ELISA in the diagnosis ofN. caninuminfection. In total, 26 aborted fetuses and 813 blood samples were collected from 8 dairy herds in Beijing (n=212) and Tianjin (n=601), China. Fifteen fetuses (57·7%) were testedN. caninum-positive by the nested PCR.N. caninumDNA was detected from the brain of 52%, kidneys of 22%, skeletal muscle of 18%, and heart of 4% of the aborted fetuses. The PCR-positive cases (55%, 11/20) were higher than seropositive cows (40%, 8/20) in a subset of 20 fetuses, but the PCR results of blood samples of the 20 cows were all negative. The seroprevalence of the 813 samples was 15·5% (43·4% of samples from Beijing, 5·7% of samples from Tianjin), compared to the PCR-positive blood samples of 0·9%. Our study showed that the nested PCR is a valuable diagnostic tool for the primary diagnosis ofN. caninumin aborted fetuses, while ELISA is the preferred assay for testing blood samples collected from cows. The two assays are complementary in determining whether abortions are associated withN. caninuminfection in cattle.

A duplex nested-PCR assay for detection of mud crab reovirus and mud crab dicistrovirus-1

2013 ◽  
Vol 20 (4) ◽  
pp. 808-815
Author(s):  
Di ZHANG ◽  
Keng YANG ◽  
Youlu SU ◽  
Juan FENG ◽  
Zhixun GUO
Keyword(s):  
Nested Pcr ◽  
Mud Crab ◽  
Pcr Assay

scholarly journals Molecular characteristics of HBV infection among blood donors tested HBsAg reactive in a single ELISA test in southern China

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xianlin Ye ◽  
Tong Li ◽  
Ran Li ◽  
Heng Liu ◽  
Junpeng Zhao ◽  
...  
Keyword(s):  
Viral Load ◽  
Hepatitis B ◽  
Nested Pcr ◽  
Blood Donors ◽  
Southern China ◽  
Core Promoter ◽  
Elisa Test ◽  
Hbv Infection ◽  
Molecular Characteristics ◽  
Blood Samples

Abstract Background Hepatitis B virus (HBV) infection is a major concern for blood safety in high-prevalence HBV countries such as China. In Shenzhen, dual hepatitis B surface antigen (HBsAg) enzyme-linked immunosorbent assays (ELISAs) have been adopted in parallel with nucleic acid testing (NAT) for donors for over a decade. A small proportion of blood donors test reactive (R) for HBsAg but negative through routine NAT, which can lead to HBV infection with an extremely low viral load. Objectives We aimed to investigate and analyze the molecular characteristics of HBV among blood donors that tested HBsAg R in a single ELISA test. Methods Blood donations were evaluated in this study if confirmed HBsAg R through one of two ELISA kits. Samples with non-reactive (NR) results by NAT were collected and tested for HBsAg by chemiluminescent microparticle immunoassay (CLIA) with a neutralization test. The level of HBsAg was further assessed by electrochemiluminescence immunoassay (ECLIA). The viral basic core promoter (BCP) and pre-core (PC) and S regions were amplified by nested PCR. Quantitative real-time PCR (qPCR) for viral load determination and individual donation (ID)-NAT were adopted simultaneously. HBsAg was confirmed with CLIA, ECLIA, nested PCR, qPCR, and ID-NAT. Results Of the 100,252 donations, 38 and 41 were identified as HBsAg R with Wantai and DiaSorin ELISA kits, respectively. Seventy-nine (0.077%, 79/100,252) blood samples with ELISA R-NR and NAT NR results were enrolled in the study. Of these, 17 (21.5%,17/79) were confirmed as HBsAg-positive. Of the 14 genotyped cases, 78.6% (11/14) were genotype B, and C and D were observed in two and one sample, respectively. Mutations were found in the S gene, including Y100C, Y103I, G145R, and L175S, which can affect the detection of HBsAg. A high-frequency mutation, T1719G (93.3%), was detected in the BCP/PC region, which reduced the viral replication. Conclusion A small number of blood samples with HBsAg ELISA R-NR and NAT NR results were confirmed as HBV infection, viral nucleic acids were found in most of the samples through routine NAT methods. It is necessary to employ more sensitive and specific assays for the detection of HBV infection among blood donors.

Development of a hemi-nested PCR assay for the specific detection ofMelissococcus pluton

1998 ◽  
Vol 37 (3) ◽  
pp. 165-174 ◽  
Author(s):  
Steven P Djordjevic ◽  
Kerrie Noone ◽  
Lisa Smith ◽  
Michael A Z Hornitzky
Keyword(s):  
Nested Pcr ◽  
Specific Detection ◽  
Pcr Assay

scholarly journals Neurological disorder in cattle associated with bovine herpesvirus 4

2011 ◽  
Vol 63 (4) ◽  
pp. 828-835 ◽  
Author(s):  
E.A. Costa ◽  
A.C. Vasconcelos ◽  
M.R.Q. Bomfim ◽  
H.B. Amorim ◽  
G.B.L. Lima ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Clinical Course ◽  
Bovine Herpesvirus ◽  
Pcr Assay ◽  
Bovine Herpesvirus 1 ◽  
Herpesvirus Type ◽  
Neurological Signs ◽  
Bovine Herpesvirus 4 ◽  
Herpesvirus 1 ◽  
Herpesvirus 4

A nested PCR assay was used to diagnose bovine encephalitis through herpesviruses including bovine herpesvirus 5 (BHV-5), bovine herpesvirus 1 (BHV-1), Aujeszky's disease virus (SHV-1), and ovine herpesvirus 2 (OHV-2) in 14 fragments of central nervous system (CNS) from cattle that died with neurological signs. In addition, as some samples of bovine herpesvirus type 4 (BHV-4) have been isolated from neural tissue, it was also tested by nested PCR. The cases of encephalitis occurred in isolation at different times of the year and did not present any seasonality. The duration of the clinical course ranged between 1 to 15 days, and in 64.3% of the cases it manifested between 1 to 2 days. The most frequently observed neurological signs were ataxia, recumbency, unsteadiness and inability to stand, opisthotonus, paddling movements, nystagmus and ptyalism. In the nested assay, there was no evidence of: BHV-1, SHV-1 or OHV-2 in the DNA obtained from the CNS in any of the samples. But the presence of BHV-4 was found in all fragments of the CNS in cattle which died presenting neurological signs. Moreover, BHV-5 was found in association with BHV-4 in two of these samples.

scholarly journals First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 353-353 ◽  
Author(s):  
M. Šeruga Musić ◽  
D. Škorić ◽  
I. Haluška ◽  
I. Križanac ◽  
J. Plavec ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Reference Strain ◽  
Simultaneous Detection ◽  
Pinot Noir ◽  
Vitis Vinifera L ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Flavescence Dorée ◽  
Scaphoideus Titanus

Flavescence dorée (FD) and Bois noir (BN) phytoplasmas are principal grapevine yellows (GY) agents in the wider Euro-Mediterranean Region. While BN phytoplasma belongs to the ribosomal subgroup 16SrXII-A, the FD agents belong either to the ribosomal subgroups 16SrV-C or -D. During the official GY survey in 2009, 40 symptomatic grapevines (Vitis vinifera L.) were sampled throughout grapevine-growing regions in Croatia. Typical GY symptoms of leaf yellowing or reddening were evident on white and red varieties, respectively. Leaf rolling as well as irregular lignification of the shoots and withering of clusters were also observed. Phloem tissue from cuttings and leaf veins from mature vines were sampled for total DNA extraction and amplification of phytoplasma 16S rRNA gene by using generic primers P1/P7 in a direct PCR assay followed by a nested PCR using primer pair R16F2n/R2 (2). Phytoplasma ribosomal group affiliation was determined by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products with enzyme Tru1I (Fermentas, Vilnius, Lithuania). These initial findings were validated and augmented by a triplex real-time PCR assay targeting the nonribosomal map gene. This assay enables simultaneous detection of BN and FD (16SrV-C and -D) phytoplasmas in grapevine (3). Assay results revealed the majority of GY positive vines (19 of 40) contained BN phytoplasma which is widespread. For the first time in Croatia, two red variety samples, Pinot Noir and Plemenka Crvena, from the vicinity of Ozalj (Vivodina) and Zagreb (Brezje), respectively, were found to harbor FD-related phytoplasmas. Fragments amplified by P1/P7 primers from latter samples were cloned and sequenced. Sequence analyses using online interactive tool iPhyClassifier (4) revealed that the phytoplasma under study from Pinot Noir sample (GenBank Accession No. HQ712064) is a member of 16SrV-C subgroup and shares 99.87% similarity with 16S rDNA sequence of the reference strain (GenBank Accession No. AF176319). The sequence from the Plemenka Crvena sample (GenBank Accession No. HQ712065) shares 99.54% similarity with the reference strain and has the most similar virtual RFLP pattern to the one of the 16SrV-C subgroup (GenBank Accession No. AY197642). These findings are currently limited to vineyards in northwestern Croatia. Even so, the presence of FD principal cicadellid vector Scaphoideus titanus in the country and the occurrence and distribution of FD in neighboring countries (1,2) are factors indicating that the spread of FD in Croatia is highly probable. References: (1) L. Filippin et al. Plant Pathol. 58:826, 2009. (2) S. Kuzmanović et al. Vitis 47:105, 2008. (3) C. Pelletier et al. Vitis 48:87, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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