Real-time PCR Biochip for On-Site Detection of Coxiella Burnetii in Ticks

Abstract Background: Q fever, a zoonosis caused by Coxiella burnetii, has adverse effects on public health. Ticks are the natural reservoirs of C. burnetii and they contribute to the transmission of the pathogen. A tool for rapid, sensitive, and accurate detection of C. burnetii from ticks is important for the prevention of Q fever. Methods: Ultra-rapid real-time PCR (UR-qPCR) as a chip-based real-time PCR system was developed for the detection of C. burnetii from ticks. The UR-qPCR system was established and evaluated for the rapidity, sensitivity, and specificity of C. burnetii detection. Results: C. burnetii was detected using UR-qPCR from 5,644 larval, nymphal, and adult ticks from 408 pools collected from livestock and epidemiologically linked environments in two provinces, Gangwon and Jeju, in Korea. Ticks from three species were identified; Haemaphysalis longicornis accounted for the highest number, present in 333 of 408 pools (81.62%), followed by Haemaphysalis flava in 62 pools (15.19%) and Ixodes nipponensis in 13 pools (3.19%). The rapidity and sensitivity of PCR detection was demonstrated with the sufficient amplification and detection of approximately 56 copies of C. burnetii DNA with only 20 min of PCR amplification. The kappa value for the diagnostic agreement between UR-qPCR and stationary qPCR was in perfect agreement (p = 1). PCR detection and sequencing indicated that C. burnetii was present in 5 of the 408 pools (1.23%), in which four pools contained H. longicornis and one pool contained H. flava. The infection rates of C. burnetii in the tick pools collected from Gangwon and Jeju Provinces were 1.70% and 0.58%, respectively. Phylogenetic analysis indicated a close relationship between the detected C. burnetii and those originated from goats, humans, and ticks in different countries, such as USA, France, Germany, and Serbia. Conclusions: The results of this study could be important for the prevention and control of Q fever in the two provinces. The UR-qPCR with its features of mobility, sensitivity, and rapidity is helpful for constructing early alert systems in the field for C. burnetii in ticks and for alleviating the transmission and economic damage due to Q fever.

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Identification of Coxiella burnetii in Raw Milk of Livestock Animal in Iran

International Journal of Microbiology ◽

10.1155/2021/6632036 ◽

2021 ◽

Vol 2021 ◽

pp. 1-5

Author(s):

Ashraf Mohabati Mobarez ◽

Ehsan Mostafavi ◽

Mohammad Khalili ◽

Saber Esmaeili

Keyword(s):

Real Time ◽

Coxiella Burnetii ◽

Real Time Pcr ◽

Q Fever ◽

Goat Milk ◽

Raw Milk ◽

Molecular Evidence ◽

Milk Samples ◽

Sheep Milk ◽

Dairy Animals

Coxiella burnetii is the causative agent of Q fever in humans and animals. This study aimed to determine the frequency of C. burnetii in milk samples of dairy animals (goats, sheep, and cattle) in some selected regions in Iran, where there is no information about prevalence of C. burnetii. In this study, 162 individual milk samples were collected from 43 farms in three provinces (Tehran, Hamadan, and Mazandaran). Real-time PCR was used for the detection of IS1111a element of C. burnetii. In total, 23 of 162 samples (14.2%, 95% conﬁdence interval (CI): 9.65–20.2%) were positive for C. burnetii by real-time PCR. C. burnetii was detected in 10.17% (95% CI: 4.74–20.46) of goat milk samples. In sheep milk samples, 18.6% (95% CI: 9.74–32.62) were positive, and C. burnetii was detected in 15% (95% CI: 8.1–26.11) of cattle milk samples. Molecular evidence of the presence of C. burnetii was seen in milk samples of dairy animals in all the studied regions. These findings demonstrated that C. burnetii infection, especially in raw milk samples, deserves more attention from the health care system and veterinary organization in Iran.

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Endpoint PCR Detection of Sars-CoV-2 RNA

10.1101/2020.07.21.20158337 ◽

2020 ◽

Cited By ~ 1

Author(s):

Samuel Elliot Moses ◽

Claire Warren ◽

Phil Robinson ◽

Jon Curtis ◽

Steve Asquith ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Pcr Amplification ◽

Positive Sample ◽

Pcr Detection ◽

Rate Limiting Step ◽

Copy Numbers ◽

Qualitative Detection ◽

Large Scale Testing

Quantitative real-time PCR methods have been used to perform approximately 278 million tests for COVID-19 up to mid-July 2020. Real-time PCR involves a rate limiting step where the samples are measured in situ during each PCR amplification cycle. This creates a bottleneck limiting scalability and as a consequence reducing access to inexpensive reliable testing at national and international scales. We investigated endpoint PCR for the qualitative detection of SARS-CoV-2 sequences on synthetic RNA standards and hospital patient samples. The endpoint PCR detection limit is constrained only by the stochastics of low copy numbers and reliably detected single copies of synthetic RNA standards. On a set of 30 patient samples, endpoint PCR found one additional positive sample and was able to confirm an indeterminate sample as negative. These results were found using 4 μl reagent and 1 μl of sample representing an 80% reduction relative to the NHS protocol (20 μl reagent and 5 μl sample). These results indicate that endpoint PCR should be the method of choice for large scale testing programmes. Based on the experience from ultra-high throughput genotyping efforts a single workflow using 384-well plates has similar PCR capacity (250 Million) to that required for all testing done worldwide during the first 7 month of the pandemic.

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Diagnosis of Acute Q Fever by Detection of Coxiella burnetii DNA using Real-Time PCR, Employing a Commercial Genesig Easy Kit

JOURNAL FOR CLINICAL AND DIAGNOSTIC RESEARCH ◽

10.7860/jcdr/2017/31005.10606 ◽

2017 ◽

Cited By ~ 2

Author(s):

Jothimani Pradeep

Keyword(s):

Real Time ◽

Coxiella Burnetii ◽

Real Time Pcr ◽

Q Fever ◽

Acute Q Fever

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Coxiella burnetii real-time PCR - IS1111a method (Q fever) v1 (protocols.io.rhhd336)

protocols.io ◽

10.17504/protocols.io.rhhd336 ◽

2018 ◽

Author(s):

Judy Northill ◽

Ian Mackay

Keyword(s):

Real Time ◽

Coxiella Burnetii ◽

Real Time Pcr ◽

Q Fever

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PREVALENCE OF COXIELLA BURNETII INFECTION IN CATTLE, BLACK BENGAL GOATS AND TICKS IN BANGLADESH

Bangladesh Journal of Veterinary Medicine ◽

10.3329/bjvm.v14i1.28827 ◽

2016 ◽

Vol 14 (1) ◽

pp. 65-68 ◽

Cited By ~ 3

Author(s):

A. Chakrabartty ◽

P. K. Bhattacharjee ◽

R. R. Sarker ◽

A. K. M. A. Rahman ◽

K. Henning ◽

...

Keyword(s):

Real Time ◽

Coxiella Burnetii ◽

Real Time Pcr ◽

Q Fever ◽

Elisa Test ◽

Quantitative Real Time Pcr ◽

Serum Samples ◽

Test Kit ◽

History Of ◽

Cattle Serum

The objectives of this study were to determine the prevalence of Coxiella burnetii infection in domestic ruminants and to detect Coxiella burnetii DNA from ticks and serum samples. A total of 24 ticks, 91 goats and 81 cattle serum samples with the history of abortion and reproductive disorders were collected from the different areas in Bangladesh. The serum samples were tested by CHEKIT Q-Fever Antibody ELISA Test Kit and Coxiella burnetii DNA was detected by multiplex quantitative real- time PCR. The overall prevalence was 7.6% and 6.1% in goats and cattle, respectively. However, none of seropositive samples and tick samples was positive in quantitative real-time PCR.

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Evaluation of the Effectiveness of Q Fever Treatment with Oxytetracycline

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/v10213-012-0090-5 ◽

2012 ◽

Vol 56 (4) ◽

pp. 513-517 ◽

Cited By ~ 2

Keyword(s):

Real Time ◽

Coxiella Burnetii ◽

Real Time Pcr ◽

Q Fever ◽

The Third ◽

Molecular Studies ◽

Fever Treatment ◽

Specific Sequences

Abstract The aim of the study was to evaluate the effectiveness of Q fever treatment with oxytetracycline based on the level of Coxiella burnetii antibodies in the sera of infected goats and cows, and excretion of Coxiella burnetii in milk. The study was performed in naturally infected goats and cattle. Forty-six goat sera and 35 cows’ sera were investigated three times before, and twice after treatment with oxytetracycline. The percentage of seronegative goats after treatment (the third examination) was 86.96% while the percentage of seronegative cows after treatment was 52.77%. Moreover, the molecular studies (real-time PCR) of cheese from milk of these animals showed that the specific sequences of DNA for Coxiella burnetii were present despite treatment with oxytetracycline.

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Faculty Opinions recommendation of Broadly targeted triplex real-time PCR detection of influenza A, B and C viruses based on the nucleoprotein gene and a novel "MegaBeacon" probe strategy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.713998087.789552802 ◽

2012 ◽

Author(s):

Sanjay Tyagi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Influenza A ◽

Pcr Detection ◽

Nucleoprotein Gene

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Persistence of SARS-CoV-2 Viral RNA in Nasopharyngeal Swabs after Death: An Observational Study

Microorganisms ◽

10.3390/microorganisms9040800 ◽

2021 ◽

Vol 9 (4) ◽

pp. 800

Author(s):

Francesca Servadei ◽

Silvestro Mauriello ◽

Manuel Scimeca ◽

Bartolo Caggiano ◽

Marco Ciotti ◽

...

Keyword(s):

Viral Load ◽

Observational Study ◽

Real Time ◽

Real Time Pcr ◽

Pcr Detection ◽

Viral Rna ◽

Clinical Documentation ◽

Post Mortem ◽

Medical Assistance ◽

Time Points

The aim of this study was to investigate the persistence of SARS-CoV-2 in post-mortem swabs of subjects who died from SARS-CoV-2 infection. The presence of the virus was evaluated post-mortem from airways of 27 SARS-CoV-2 positive patients at three different time points (T1 2 h; T2 12 h; T3 24 h) by real-time PCR. Detection of antibodies to SARS-CoV-2 was performed by Maglumi 2019-nCoV IgM/IgG chemiluminescence assay. SARS-CoV-2 viral RNA was still detectable in 70.3% of cases within 2 h after death and in 66,6% of cases up to 24 h after death. Our data showed an increase of the viral load in 78,6% of positive individuals 24 h post-mortem (T3) in comparison to that evaluated 2 h after death (T1). Noteworthy, we detected a positive T3 post-mortem swab (24 h after death) from 4 subjects who were negative at T1 (2 h after death). The results of our study may have an important value in the management of deceased subjects not only with a suspected or confirmed diagnosis of SARS-CoV-2, but also for unspecified causes and in the absence of clinical documentation or medical assistance.

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