A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay

Lockhart, Michelle G.; Graves, Stephen; Banazis, Michael; Fenwick, Stan; Stenos, John

doi:10.1111/j.1472-765x.2011.03034.x

Cited by 33 publications

(29 citation statements)

References 21 publications

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“…() and Lockhart et al. (). Briefly, each reaction mix consisted of 12.5 μL Quantitect Probe PCR Mix (QIAGEN), 10 pmol of each COM1 primers F‐AAA ACC TCC GCG TTG TCT TCA and R‐GCT AAT GAT ACT TTG GCA GCG TAT TG or IS1111 primers F‐GTC TTA AGG TGG GCT GCG TG and R‐CCC CGA ATC TCA TTG ATC AGC, 5 pmol of probe COM1 FAM‐AGA ACT GCC CAT TTT TGG CGG CCA‐BHQ1 or IS1111 probe FAM‐AGC GAA CCA TTG GTA TCG GAC GTT TAT GG‐BHQ1, and 5 μL of template DNA in a final reaction volume of 25 μL.…”

Section: Methodsmentioning

confidence: 99%

Potential Animal and Environmental Sources of Q Fever Infection for Humans in Queensland

Tozer

Lambert

Strong

et al. 2013

Zoonoses and Public Health

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Q fever is a vaccine-preventable disease; despite this, high annual notification numbers are still recorded in Australia. We have previously shown seroprevalence in Queensland metropolitan regions is approaching that of rural areas. This study investigated the presence of nucleic acid from Coxiella burnetii, the agent responsible for Q fever, in a number of animal and environmental samples collected throughout Queensland, to identify potential sources of human infection. Samples were collected from 129 geographical locations and included urine, faeces and whole blood from 22 different animal species; 45 ticks were removed from two species, canines and possums; 151 soil samples; 72 atmospheric dust samples collected from two locations and 50 dust swabs collected from domestic vacuum cleaners. PCR testing was performed targeting the IS1111 and COM1 genes for the specific detection of C. burnetii DNA. There were 85 detections from 1318 animal samples, giving a detection rate for each sample type ranging from 2.1 to 6.8%. Equine samples produced a detection rate of 11.9%, whilst feline and canine samples showed detection rates of 7.8% and 5.2%, respectively. Native animals had varying detection rates: pooled urines from flying foxes had 7.8%, whilst koalas had 5.1%, and 6.7% of ticks screened were positive. The soil and dust samples showed the presence of C. burnetii DNA ranging from 2.0 to 6.9%, respectively. These data show that specimens from a variety of animal species and the general environment provide a number of potential sources for C. burnetii infections of humans living in Queensland. These previously unrecognized sources may account for the high seroprevalence rates seen in putative low-risk communities, including Q fever patients with no direct animal contact and those subjects living in a low-risk urban environment.

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Section: Methodsmentioning

confidence: 99%

Potential Animal and Environmental Sources of Q Fever Infection for Humans in Queensland

Tozer

Lambert

Strong

et al. 2013

Zoonoses and Public Health

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“…For quantification of C. burnetii specific DNA, a Com1 qPCR was used. 22 The C t values of positive qPCR samples were used to calculate the approximate copy numbers/genome equivalent in each dilution of inoculum and in each homogenized mice spleen.…”

Section: Methodsmentioning

confidence: 99%

The Attenuated Nine Mile Phase II Clone 4/RSA439 Strain of Coxiella burnetii is Highly Virulent for Severe Combined Immunodeficient (SCID) Mice

Islam¹,

Lockhart²,

Stenos³

et al. 2013

The American Journal of Tropical Medicine and Hygiene

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Abstract. The Nine Mile phase II clone 4 (NMIIC4) strain of Coxiella burnetii is an attenuated phase II strain that has lost the genes for virulence determinant type 1 lipopolysaccharide. These bacteria were very virulent for severe combined immunodeficient (SCID) mice. The lethal dose 50 (LD 50 ) was~10 bacteria. Infected SCID mice died between Day 28 and Day 53 post-infection. At termination of the experiment (Day 60) only 5 of 24 mice had survived. The degree of splenomegaly was directly related to the bacterial load in the SCID mice spleens. The NMIIC4 was avirulent in immunocompetent wild mice and bacterial DNA copies in splenic tissue were extremely low. The SCID mice that were inoculated with high doses of heat inactivated NMIIC4 C. burnetii were all alive at Day 60 and without splenomegaly. It appears that the phase I lipopolysaccharide present in virulent Nine Mile phase I but not in attenuated NMIIC4 is not the only virulence factor for C. burnetii. BACKGROUND

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“…qPCR DNA was extracted from 100 lL of each dilution (cell culture and SCID mice spleen homogenates) using the Qiagen Extraction Kit (Qiagen, Germany) following the manufacturers instructions. Each 50-lL eluant was analysed with our in-house C. burnetii specific com1 qPCR assay (Lockhart et al, 2011). As the DNA was eluted into a volume of 50 lL and of which 5 lL was analysed in each reaction, this was effectively one-tenth of the bacterial DNA in the original starting suspension.…”

Section: Coxiella Burnetii Isolatesmentioning

confidence: 99%

Detecting and measuring small numbers of viableCoxiella burnetii

Lockhart¹,

Islam

Graves

et al. 2012

FEMS Immunol Med Microbiol

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Coxiella burnetii is an acidophilic, intracellular bacterium that causes the human disease Q fever. In some studies, it is important to distinguish between viable and nonviable C. burnetii. We compared four methods for detecting and measuring viable C. burnetii in biological samples as follows: growth in two different cell culture lines, infection of severe combined immunodeficient (SCID) mice (leading to death) and infection of SCID mice with detection of C. burnetii in their spleen (after euthanasia at day 50 postinfection). Two isolates of C. burnetii were used ('Henzerling' and 'Arandale'). Our in-house qPCR assay for C. burnetii DNA was used as a control. SCID mouse inoculation was more sensitive than cell culture. The assay that detected C. burnetii in SCID mouse spleens was slightly more sensitive than SCID mice deaths alone. Approximately one viable C. burnetii cell could be detected by this method, making it suitable for determining the viability of C. burnetii in a sample.

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A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay

Cited by 33 publications

References 21 publications

Potential Animal and Environmental Sources of Q Fever Infection for Humans in Queensland

Potential Animal and Environmental Sources of Q Fever Infection for Humans in Queensland

The Attenuated Nine Mile Phase II Clone 4/RSA439 Strain of Coxiella burnetii is Highly Virulent for Severe Combined Immunodeficient (SCID) Mice

Detecting and measuring small numbers of viableCoxiella burnetii

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