Detection of viral DNA of myxoma virus using a validated PCR method with an internal amplification control

A novel RPA–IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 103CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA–IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

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INVESTIGATION OF THE FREQUENCY, GENOTYPE DISTRIBUTION AND PROBABLE RISK FACTORS OF HUMAN PAPILLOMAVIRUS (HPV) AMONG WOMENS ATTENDING TO FAMILY HEALTH CENTER

Gevher Nesibe Journal, IESDR ◽

10.46648/gnj.173 ◽

2021 ◽

Vol 6 (11) ◽

pp. 12-19

Author(s):

Meryem ÇOLAK ◽

Merve KARTAL DEMİR

Keyword(s):

Risk Factors ◽

Health Center ◽

Sexual Intercourse ◽

Hpv Vaccine ◽

Family Health ◽

Genotype Distribution ◽

Viral Dna ◽

Hpv Dna ◽

First Sexual Intercourse ◽

Pcr Method

It is important to understand the frequency of Human Papilloma Virus (HPV) and the factors associated with HPV infections, to determine prevention, control and measures to be taken. In this study, it was aimed to investigate the HPV prevalence, genotype distribution and possible risk factors in women applying to the Family Health Center. Sexually active, clinically asymptomatic fifty women aged 30-65 who applied to Safranbolu Family Health Center between October 2018 and March 2019 were included in the study. In order to compare HPV-DNA positivity and socio-demographic characteristics, the education status, number of pregnancies, age of first sexual intercourse, presence of chronic disease, pap smear test and HPV vaccine status, etc. information was recorded and cervical swab samples were taken for detection of HPV-DNA. Viral DNA was isolated with the QIAamp® Viral DNA Kit (QIAGEN, Germany) according to the manufacturer's protocol. The presence of HPV-DNA was investigated using the MY09 / MY11 primers by the Polymerase Chain Reaction (PCR) method, positive samples were amplified using type-specific primers by PCR method and HPV typing was performed. HPV-DNA positivity was detected as 6% (3/50) in the study. Two samples were typed as HPV16; and one samples was typed as HPV31. It was observed that all HPV DNA positive samples were high risk oncogenic HPV types. When the probable risk factors are examined; It was observed that the first sexual intercourse age of patients was 16-21 years, and the number of pregnancies was ≥3 among the HPV-DNA positive women. A statistically significant relation was found with HPV-DNA positivity between the age of first sexual intercourse and the number of pregnancies (p <0.05). HPV-DNA positivity was not observed in any woman protected by a condom, which is considered to be the safest in terms of to protect sexualy transmission infections. It was observed that any women participating in the study knew the availability of HPV vaccine and did not get the HPV vaccine. It is necessary to perform studies showing the prevalence of HPV and common genotypes in a region at certain time intervals; it is thought that the results will guide cervical cancer prevention and vaccination.

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Safety of an Oncolytic Myxoma Virus in Dogs with Soft Tissue Sarcoma

Viruses ◽

10.3390/v10080398 ◽

2018 ◽

Vol 10 (8) ◽

pp. 398 ◽

Cited By ~ 10

Author(s):

Amy MacNeill ◽

Kristen Weishaar ◽

Bernard Séguin ◽

Barbara Powers

Keyword(s):

Soft Tissue ◽

Human Cancer ◽

Treatment Options ◽

Safety Information ◽

Soft Tissue Sarcomas ◽

Myxoma Virus ◽

Oncolytic Viruses ◽

Multiple Time ◽

Viral Dna ◽

Multiple Time Points

Many oncolytic viruses that are efficacious in murine cancer models are ineffective in humans. The outcomes of oncolytic virus treatment in dogs with spontaneous tumors may better predict human cancer response and improve treatment options for dogs with cancer. The objectives of this study were to evaluate the safety of treatment with myxoma virus lacking the serp2 gene (MYXVΔserp2) and determine its immunogenicity in dogs. To achieve these objectives, dogs with spontaneous soft tissue sarcomas were treated with MYXVΔserp2 intratumorally (n = 5) or post-operatively (n = 5). In dogs treated intratumorally, clinical scores were recorded and tumor biopsies and swabs (from the mouth and virus injection site) were analyzed for viral DNA at multiple time-points. In all dogs, blood, urine, and feces were frequently collected to evaluate organ function, virus distribution, and immune response. No detrimental effects of MYXVΔserp2 treatment were observed in any canine cancer patients. No clinically significant changes in complete blood profiles, serum chemistry analyses, or urinalyses were measured. Viral DNA was isolated from one tumor swab, but viral dissemination was not observed. Anti-MYXV antibodies were occasionally detected. These findings provide needed safety information to advance clinical trials using MYXVΔserp2 to treat patients with cancer.

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Evaluation of Most-Probable-Number–PCR Method with Internal Amplification Control for the Counting of Total and Pathogenic Vibrio parahaemolyticus in Frozen Shrimps

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-165 ◽

2012 ◽

Vol 75 (1) ◽

pp. 150-153 ◽

Cited By ~ 12

Author(s):

S. COPIN ◽

A. ROBERT-PILLOT ◽

P. MALLE ◽

M. L. QUILICI ◽

M. GAY

Keyword(s):

Vibrio Parahaemolyticus ◽

Direct Detection ◽

Selective Medium ◽

Internal Amplification Control ◽

Most Probable Number ◽

Probable Number ◽

Pathogenic Vibrio ◽

Biochemical Identification ◽

Pcr Method ◽

Frozen Shrimp

The most-probable-number (MPN) method is often time-consuming for the isolation, detection, and quantification of Vibrio parahaemolyticus from natural sources. MPN counting of V. parahaemolyticus bacteria usually involves the isolation of typical V. parahaemolyticus colonies on selective medium, with subsequent confirmation by biochemical identification. In this study, we evaluated the use of a PCR on MPN enrichment cultures (MPN-PCR) for the direct detection of total and pathogenic V. parahaemolyticus cells in frozen shrimp. This reaction targeted the R72H, tdh, and trh sequences. An internal amplification control was added to the samples before R72H amplification. There was an excellent correlation between the results of the two methods for artificially inoculated and natural shrimp samples. Of 36 natural samples, 28 tested positive for the presence of V. parahaemolyticus, with an MPN value of 2 × 10−1 to 9.2 × 101 per g. No pathogenic V. parahaemolyticus cells were detected. The test had a detection limit of one V. parahaemolyticus organism per g and was completed within two working days. These results support the use of the combination of PCR with MPN for the detection of total or potentially pathogenic V. parahaemolyticus cells in frozen shrimp.

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TaqMan-Based Real-Time PCR Method for Detection of Yersinia pseudotuberculosis in Food

Applied and Environmental Microbiology ◽

10.1128/aem.01459-08 ◽

2008 ◽

Vol 74 (20) ◽

pp. 6465-6469 ◽

Cited By ~ 28

Author(s):

S. Thisted Lambertz ◽

C. Nilsson ◽

S. Hallanvuo

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Yersinia Pseudotuberculosis ◽

Internal Amplification Control ◽

Food Items ◽

Specific Assay ◽

Food Borne ◽

Pcr Method ◽

Probe Set

ABSTRACT A sensitive and specific assay for detection of food-borne pathogenic Yersinia pseudotuberculosis was developed. The primer-probe set was designed to target a 157-bp sequence of the chromosomally located gene ail. The complete method, including an internal amplification control, was evaluated for several different food items.

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A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids

International Journal of Molecular Sciences ◽

10.3390/ijms222112052 ◽

2021 ◽

Vol 22 (21) ◽

pp. 12052

Author(s):

Fábio A. Abade dos Santos ◽

Carina L. Carvalho ◽

Francisco Parra ◽

Kevin P. Dalton ◽

Maria C. Peleteiro ◽

...

Keyword(s):

Real Time ◽

Housekeeping Gene ◽

Myxoma Virus ◽

European Rabbit ◽

Fast Method ◽

Wild Rabbit ◽

Viral Dna ◽

Eukaryotic Organisms ◽

Virus Strains ◽

Iberian Hare

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

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Detection of peanut (Arachis hypogaea) allergen by Real-time PCR method with internal amplification control

Food Chemistry ◽

10.1016/j.foodchem.2014.11.091 ◽

2015 ◽

Vol 174 ◽

pp. 547-552 ◽

Cited By ~ 24

Author(s):

Wen-Ju Zhang ◽

Qin Cai ◽

Xiao Guan ◽

Qin Chen

Keyword(s):

Real Time ◽

Arachis Hypogaea ◽

Real Time Pcr ◽

Internal Amplification Control ◽

Pcr Method

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Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods

Food Microbiology ◽

10.1016/j.fm.2012.08.001 ◽

2012 ◽

Vol 32 (2) ◽

pp. 397-405 ◽

Cited By ~ 13

Author(s):

Alicia Rodríguez ◽

María L. Werning ◽

Mar Rodríguez ◽

Elena Bermúdez ◽

Juan J. Córdoba

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Cyclopiazonic Acid ◽

Internal Amplification Control ◽

Quantitative Real Time Pcr ◽

Pcr Method

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Factors contributing to salivary human immunodeficiency virus type-1 levels measured by a Poisson distribution-based PCR method

Journal of International Medical Research ◽

10.1177/0300060517728652 ◽

2017 ◽

Vol 46 (3) ◽

pp. 996-1007 ◽

Cited By ~ 2

Author(s):

Ryo Ikeno ◽

Eiko Yamada ◽

Sayaka Yamazaki ◽

Tomoyuki Ueda ◽

Masaki Nagata ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Viral Load ◽

Oral Cavity ◽

Poisson Distribution ◽

Plasma Viral Load ◽

Viral Dna ◽

Immunodeficiency Virus ◽

Pcr Method ◽

Hiv 1

Objective To elucidate the mechanism underlying secretion of human immunodeficiency virus type 1 (HIV-1) into the oral cavity, by examining the relationships between various oral and systemic factors and the viral load in saliva. Methods Plasma and saliva samples from HIV-1 infected patients were assayed using the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, version 1.0 and a Poisson distribution-based polymerase chain reaction (PCR) method for quantifying HIV-1 RNA and DNA. Results Forty-four pairs of samples were obtained from 18 patients. Salivary viral load was approximately 10% of the plasma viral load, but higher than the plasma load in two patients. The salivary viral DNA load was < 1% of the total HIV-1 nucleic acid load except in one patient who had more viral DNA than RNA. Multiple regression analysis showed that salivary viral load was significantly correlated with plasma viral load (partial correlation coefficient, 0.90) and the community periodontal index (–0.63). Conclusions The present results suggest that excretion through salivary glands, but not occult bleeding, may be a major pathway of HIV-1 into the oral cavity.

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Slovenska priporočila pri okužbi s parvovirusom B19 v nosečnosti

Slovenian Medical Journal ◽

10.6016/zdravvestn.2618 ◽

2018 ◽

Vol 87 (1-2) ◽

Author(s):

Nina Osvald Avguštin ◽

Tanja Premru Sršen ◽

Barbara Šajina Stritar ◽

Faris Mujezinović

Keyword(s):

Pregnant Women ◽

Parvovirus B19 ◽

Severe Anaemia ◽

Ultrasound Screening ◽

Viral Dna ◽

Erythema Infectiosum ◽

Mild Disease ◽

Tertiary Centre ◽

Fetal Infection ◽

Pcr Method

Parvovirus B19 (B19V) causes a mild disease called erythema infectiosum, also known as the fifh disease that aﬀects mostly children and young adults. The virus can be transferred to the fetus during pregnancy in 31 to 51 % of the cases and can cause severe anaemia, non-immune hydrops fetalis or fetal death due to inhibition of erythropoiesis. It also aﬀects the heart muscle, central nervous system, bones, and most likely can cause a subsequent arrest in children’s neurological development. It is estimated that 25–45 % of pregnant women are seronegative with a high risk of infection during pregnancy. A B19V infection in pregnant women is determined by detecting specific IgM and IgG antibodies, and in case of doubt, by using PCR method to detect viral DNA. Fetal infection with B19V is confirmed by detecting viral DNA in the amniotic fluid. In the case of either a suspected or confirmed fetal infection we monitor the fetus by ultrasound screening in a tertiary centre. We treat the fetus with an intrauterine transfusion at the first signs of anaemia or hydrops. To prevent fresh infections with B19V during pregnancy we should raise awareness amongst women and healthcare workers about the risks it poses for the fetus. The recommendations for management of women who are exposed to, are at risk of developing, or have developed B19V infection in pregnancy are published in this article.

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