Sensitivity of nested PCR toward human cytomegalovirus-DNA in native sera and in extracted DNA of serum samples

Abstract Objective This study aimed to investigate characteristics of human cytomegalovirus (HCMV) glycoprotein H (gH) genotypes in urine, throat swab, and serum from children and breast milk from children's mothers. Methods Fresh urine samples, throat swabs, or serum samples from children and breast milk samples from children's mothers were collected for HCMV DNA detection. The positive samples of HCMV DNA were further detected by fluorescent quantitative polymerase chain reaction (PCR) with gH typing. Results Of 1,703 HCMV DNA-positive samples, the highest proportion (83.3%, 85/102) of children aged between 21 days and 3 months was detected positive in breast milk samples (p = 0.002), and the highest proportion (70.5%, 110/156) of children aged above 3 months was detected positive in throat swab samples (p = 0.002). HCMV in throat swab specimens is mainly high copy (p < 0.0001), and low-copy HCMV is prevalent in breast specimens (p < 0.0001). Among them, 1,059 samples were identified as gH1 genotype, 530 samples were gH2, and 114 samples were coinfection (gH1/2). There had the highest gH2 rates (32.3%) and lowest gH1 (61.0%) rates in urine samples (p = 0.041), whereas the highest gH1 rates (71.6%) and lowest gH2 rates (19.6%) were found in breast milk samples (p = 0.032). Concerning age groups, patients aged between 21 days and 3 months had the highest gH1 proportion (p = 0.017), while patients aged above 3 months had the highest gH1 and gH2 HCMV coinfection proportion (p = 0.002). Among 43 pairs of maternal and child samples corresponding to positive samples, gH genotype of 35 pairs of samples was consistent with a rate of 81.4%. Conclusion gH1 is the predominant genotype of HCMV in each kind of sample in China. However, the distribution of the HCMV gH genotype is different among different samples.

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Different expression pattern of human cytomegalovirus-encoded microRNAs in circulation from virus latency to reactivation

Journal of Translational Medicine ◽

10.1186/s12967-020-02653-w ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Wanqing Zhou ◽

Cheng Wang ◽

Meng Ding ◽

Yuying Bian ◽

Yujie Zhong ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Characteristic Curve ◽

Antiviral Treatment ◽

Serum Levels ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Prediction Ability ◽

Stem Loop ◽

Virus Latency ◽

Polymerase Chain

Abstract Background Human cytomegalovirus (HCMV) is a beta-hersvirinae that has a high latent infection rate worldwide and can cause serious consequences in immunocompromised patients when reactivation; however, the mechanism of how HCMV convert from latent to reactivation has rarely been investigated. In the present study, we aimed to perform a comprehensive analysis of the HCMV-encoded microRNA (miRNA) profile in serum of patients upon HCMV reactivation from latency and to further evaluate its clinical significance for the disease monitoring and preventing usefulness. Methods Serum samples from 59 viremia patients and 60 age-gender matched controls were enrolled in this study for screening and validation of different expression of HCMV miRNAs. Serum concentrations of 22 known HCMV miRNAs were determined by a hydrolysis probe-based stem-loop quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. HCMV DNA was measured by quantitative real-time PCR (qPCR) with the whole blood sample. Serum HCMV IgG and IgM were assessed using enzyme linked immunosorbent assay (ELISA). Another 47 samples from 5 patients at different time points were collected to evaluate the monitoring effectiveness and disease prediction ability of differential expression HCMV-miRNAs during the antiviral treatment. Results The RT-qPCR analysis revealed that the serum levels of 16 of the 22 examined HCMV miRNAs were elevated in HCMV viremia patients compared with controls, and a profile of 8 HCMV miRNAs including hcmv-miR-US25-2-3p, hcmv-miR-US4-5p, hcmv-miR-US25-2-5p, hcmv-miR-US25-1-3p, hcmv-miR-US25-1, hcmv-miR-UL36, hcmv-miR-UL148D, hcmv-miR-US29-3p were markedly elevated (fold change > 2, P < 0.01). Receiver operating characteristic curve (ROC) analysis were performed on the selected HCMV-miRNAs in all of the patients and controls that enrolled in this study, and which ranged from 0.72 to 0.80 in the autoimmune patients. In addition, hcmv-miR-US25-1-3p levels were significantly correlated with HCMV DNA load (r = 0.349, P = 0.007), and were obviously higher in the reactivation set than the latency set in the autoimmune patients, which could be a predictor for the monitoring of the antiviral treatment. Conclusions HCMV miRNAs profile showed markedly shift-switch from latency to reactivation in circulation from HCMV infected patients and hcmv-miR-US25-1-3p may be served as a predictor for the switch upon reactivation from latency in patients suffered with autoimmune diseases.

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Application of nested PCR to detectPenicillium marneffeiin serum samples

Medical Mycology ◽

10.1080/13693780802484875 ◽

2009 ◽

Vol 47 (5) ◽

pp. 549-553 ◽

Cited By ~ 10

Author(s):

Monsicha Pongpom ◽

Thira Sirisanthana ◽

Nongnuch Vanittanakom

Keyword(s):

Nested Pcr ◽

Serum Samples

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Clinical significance of qualitative human cytomegalovirus (HCMV) detection in cell-free serum samples in HIV-infected patients at risk for HCMV disease

AIDS ◽

10.1097/00002030-199709000-00023 ◽

1997 ◽

Vol 11 (9) ◽

pp. 1195-1196

Author(s):

T. Laue ◽

T. Mertenskötter ◽

T. Grewing ◽

O. Degen ◽

J. van Lunzen ◽

...

Keyword(s):

At Risk ◽

Clinical Significance ◽

Human Cytomegalovirus ◽

Serum Samples ◽

Hcmv Disease ◽

Patients At Risk ◽

Free Serum

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Detection of avian oncogenic Marek’s disease herpesvirus DNA in human sera

Journal of General Virology ◽

10.1099/0022-1317-82-1-233 ◽

2001 ◽

Vol 82 (1) ◽

pp. 233-240 ◽

Cited By ~ 9

Author(s):

S. Laurent ◽

E. Esnault ◽

G. Dambrine ◽

A. Goudeau ◽

D. Choudat ◽

...

Keyword(s):

Nested Pcr ◽

Hot Spot ◽

Health Hazard ◽

Single Copy ◽

Marek's Disease ◽

Target Sequence ◽

Marek’S Disease ◽

Serum Samples ◽

Worldwide Distribution ◽

Human Sera

The avian herpesvirus Marek’s disease virus (MDV) has a worldwide distribution and is responsible for T-lymphoma in chickens. The question as to whether MDV poses a public health hazard to humans was first raised when the virus was isolated in 1967. However, no irrefutable results have been obtained in immunological and virological studies. We used a nested-PCR to detect MDV DNA in human serum samples. A total of 202 serum samples from individuals exposed and not exposed to poultry was tested by nested-PCR for a target sequence located in the MDV gD gene. The assay system was specific and sensitive, making it possible to detect a single copy of the target sequence. Forty-one (20%) of the 202 serum samples tested positive for MDV DNA. The prevalence of MDV DNA was not significantly different in the group exposed to poultry and the group not exposed to poultry. There was also no difference due to age or sex. Alignment of the 41 gD sequences amplified from human sera with eight gD sequences amplified from MDV-infected chicken sera showed a maximum nucleotide divergence of 1·65%. However, four ‘hot-spot’ mutation sites were identified, defining four groups. Interestingly, two groups contained only human MDV-gD sequences. The status of the MDV genome detected in human blood is discussed.

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Outbreak of caprine abortion by Toxoplasma gondii in Midwest Brazil

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2011001100001 ◽

2011 ◽

Vol 31 (11) ◽

pp. 933-937 ◽

Cited By ~ 6

Author(s):

Flávio Henrique Bravim Caldeira ◽

Daniel Guimarães Ubiali ◽

Isabela de Godoy ◽

Valéria Dutra ◽

Daniel Moura de Aguiar ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Brucella Abortus ◽

Nested Pcr ◽

Neospora Caninum ◽

Pcr Assay ◽

Serum Samples ◽

Pathology Laboratory ◽

Mato Grosso ◽

Veterinary Pathology ◽

Gross Lesions

An outbreak of abortion by Toxoplasma gondii in goats on a farm in the Brazilian Midwest is reported. Gross lesions were not observed in seven aborted fetuses submitted to the Veterinary Pathology Laboratory, Federal University of Mato Grosso, for necropsy investigation. The main histologic lesions were mononuclear cell pneumonia and necrotizing encephalitis in varying degrees of intensity. PCR for Brucella abortus and Neospora caninum and aerobic cultures were negative in all cases. Antibody titles against T. gondii varying from 1:1024 to 1:32.768 were detected in serum samples from four aborted goats. Nested-PCR assay for T. gondii were positive in brain samples of all cases submitted. These findings indicate that T. gondii infection should be considered in the diagnosis of abortion in goats in Midwest Brazil.

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Detection and Molecular Characterization of Orientia tsutsugamushi from Suspected Scrub Typhus Patients in Mizoram, India

International Journal of Current Microbiology and Applied Sciences ◽

10.20546/ijcmas.2021.1010.061 ◽

2021 ◽

Vol 10 (10) ◽

pp. 514-523

Author(s):

Vanramliana Gabriel Rosangkima ◽

Lalnunnemi Ralte Lalremruata ◽

Christine Vanlalbiakdiki Sailo Hunropuia ◽

Deborah Lalnghakmawii Lalfakzuala Pautu

Keyword(s):

Nested Pcr ◽

Scrub Typhus ◽

Specific Antigen ◽

Serum Samples ◽

Antigen Gene ◽

Molecular Tests ◽

Related Genotype ◽

High Degree ◽

Higher Sensitivity

Serologic and molecular tests were performed for the diagnosis and to detect O. tsutsugamushi genotypes that are circulating in the state of Mizoram, India. Blood samples from scrub typhus-suspected patients were collected from Synod Hospital, Durtlang, Mizoram. Weil-Felix and immunochromatographic test (ICT) were performed from the serum samples. Nested PCR (nPCR) amplification of 47kDa outer membrane protein antigen gene and 56kDa type-specific antigen gene were done from the whole blood. 141/177 (79.66%) and 134/177 (75.7%) cases showed the presence of antibody against scrub typhus by Weil-Felix and ICT assays respectively. 76/177 (42.93%) patients showed the presence of 47kDa OMP antigen gene by nPCR while 55/177 (31.07%) showed the presence of 56kDa TSA gene by nPCR. Phylogenetic analysis of 56kDa TSA gene sequence revealed that Karp-related genotype was the most common genotype in the study area followed by Kato-related genotype. In this study, a high degree of diversity of O. tsutsugamushi was observed similar to the observations reported from other parts of India. Nested PCR of 47kDa OMP antigen gene showed higher sensitivity as compared to nPCR amplification of 56kDa TSA gene suggesting it as the assay of choice for diagnosis of scrub typhus disease.

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Laboratory Diagnosis of Toscana Virus Infection by Enzyme Immunoassay with Recombinant Viral Nucleoprotein

Journal of Clinical Microbiology ◽

10.1128/jcm.37.3.649-652.1999 ◽

1999 ◽

Vol 37 (3) ◽

pp. 649-652 ◽

Cited By ~ 26

Author(s):

Dario Soldateschi ◽

Grazia Maria Dal Maso ◽

Marcello Valassina ◽

Laura Santini ◽

Silvia Bianchi ◽

...

Keyword(s):

Enzyme Immunoassay ◽

Nested Pcr ◽

Immune Status ◽

Laboratory Diagnosis ◽

Summer Season ◽

Immunoglobulin M ◽

Toscana Virus ◽

Serum Samples ◽

Rna Sequences ◽

Specific Serum

A recombinant enzyme immunoassay (rEIA) to detect serum immunoglobulin M (IgM) and IgG to Toscana virus (TOSV) was developed with the aim of establishing a simple and easily available assay for diagnosing acute and/or previous infections. The rEIA, based on the recombinant nucleoprotein of TOSV expressed in Escherichia coli, was evaluated with 97 serum samples collected in an area where TOSV is endemic and compared to an analogous assay based on cell-derived TOSV. Discordant results were resolved by immunoblotting (IB). Twenty-two of these samples, obtained from subjects hospitalized during the summer season with meningitis of suspected TOSV etiology, were further characterized by indirect immunofluorescence and IB, and detection of specific TOSV RNA sequences in the cerebrospinal fluid of these patients was attempted by nested PCR. The results indicated that rEIA was able to diagnose acute TOSV infection by detection of specific serum IgM in all of the subjects with TOSV meningitis confirmed by nested PCR or serology. The overall sensitivity and specificity of rEIA were both 100% for IgM detection and 100 and 96.6%, respectively, for IgG detection. Thus, rEIA appears to be a simple and reliable laboratory test for the diagnosis of acute TOSV infection and for the assessment of immune status.

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Nested PCR Assay for Detection of Granulocytic Ehrlichiae

Journal of Clinical Microbiology ◽

10.1128/jcm.36.4.1090-1095.1998 ◽

1998 ◽

Vol 36 (4) ◽

pp. 1090-1095 ◽

Cited By ~ 237

Author(s):

Robert F. Massung ◽

Kim Slater ◽

Jessica H. Owens ◽

William L. Nicholson ◽

Thomas N. Mather ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Nested Pcr ◽

Limit Of Detection ◽

Rrna Gene ◽

Pcr Assay ◽

Serum Samples ◽

Blood Specimens ◽

The 16S Rrna Gene ◽

Edta Blood

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted fromIxodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.

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A Custom-Designed Recombinant Multiepitope Protein for Human Cytomegalovirus Diagnosis

Recent Patents on Biotechnology ◽

10.2174/1872208313666190716093911 ◽

2019 ◽

Vol 13 (4) ◽

pp. 316-328

Author(s):

Patrícia A.F. Ribeiro ◽

Marilen Q. Souza ◽

Daniel S. Dias ◽

Alice C. M. Álvares ◽

Laís M. Nogueira ◽

...

Keyword(s):

Circular Dichroism ◽

Recombinant Protein ◽

Human Cytomegalovirus ◽

De Novo ◽

Structural Data ◽

Elisa Test ◽

World Population ◽

Serum Samples ◽

Serological Tests ◽

Circular Dichroïsm

Background: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. Objective: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. Methods: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). Results: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. Conclusion: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.

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