Rapid quantification of hepatitis B virus DNA by direct real-time PCR from serum without DNA extraction

Cheng, Ziqiang; Hu, Lihua; Fu, Wen-Rong; Li, Yirong

doi:10.1099/jmm.0.47154-0

Cited by 13 publications

(8 citation statements)

References 13 publications

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“…During the febrile phase, dengue viremia has been shown to range from 10 2 to 10 7 PFU/mL depending on serotype and time after onset. 14,19 Our own data is in broad agreement with these numbers, and nearly 96% of all dengue clinical samples fell within this range ( Figure 2B). In the late acute phase serum, viremia may drop to below 10 −1 PFU/ mL 14 , below the detection limit of this assay or other direct detection methods such as virus isolation.…”

Section: Discussionsupporting

confidence: 80%

“…Viral load has been correlated with the severity of clinical manifestation of dengue. 19 A correlation between viral titer in tissue culture (expressed in PFU/mL), and the limit of detection is particularly important to predict the in vivo biologic sensitivity of the assay. Therefore, we generated a standard curve to compare known virus stock concentrations to the Ct value of the qRT-PCR reaction.…”

Section: Discussionmentioning

confidence: 99%

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A Dry-format Field-deployable Quantitative Reverse Transcriptase-polymerase Chain Reaction Assay for Diagnosis of Dengue Infections

Pal²,

Ekanayake³

et al. 2008

The American Journal of Tropical Medicine and Hygiene

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We have systematically evaluated a dry-format, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay developed by Tetracore Inc. for the Cepheid SmartCycler platform to facilitate rapid diagnosis of dengue virus infections. A panel of related flaviviruses was used to evaluate the clinical specificity of the assay, and it was found to be specific to dengue. Eighty-one clinical samples previously confirmed dengue positive by virus isolation, along with 25 dengue negative control specimens were used to validate this new diagnostic assay. Using these clinical samples, the assay exhibited 98.77% sensitivity and 100% specificity. Over 85% of the clinical specimen exhibited viral loads ranging from 10(3) to 10(7) plaque-forming units per milliliter (PFU/mL). In addition, this dry-format assay is stable at ambient temperatures and requires minimal technical expertise to perform in a small thermocycler platform. These characteristics make it a promising candidate for diagnosis of dengue in mobile laboratories in the field.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

A Dry-format Field-deployable Quantitative Reverse Transcriptase-polymerase Chain Reaction Assay for Diagnosis of Dengue Infections

Pal²,

Ekanayake³

et al. 2008

The American Journal of Tropical Medicine and Hygiene

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“…Direct quantification on either dPCR platform did not appear to be suitable for the quantification of HCMV from human plasma due to partial or complete PCR inhibition that resulted in underestimation of DNA copy numbers. However, successful qPCR-based quantification of HCMV from urine samples and hepatitis B from human serum have indicated the potential suitability of direct quantification of HCMV and other viruses from these two clinical matrices [ 13 , 14 ].…”

Section: Discussionmentioning

confidence: 99%

Digital PCR for direct quantification of viruses without DNA extraction

2015

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DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.

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“…In previous studies, some researchers have reported the amplification of DNA directly from whole blood using PCR methods for diagnosing bacterial or viral infections, including Mycoplasma haemofelis [ 16 ], Bartonella quintana [ 12 ] and hepatitis B virus [ 4 ]. Moreover, in another report, PCR-DB was used for mutation screening of G M1 gangliosidosis in dogs, demonstrating that this technique can also be a good method for hereditary disease screening and not only for detecting infectious diseases [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

Direct polymerase chain reaction from blood and tissue samples for rapid diagnosis of bovine leukemia virus infection

Nishimori

Konnai

Ikebuchi

et al. 2016

The Journal of Veterinary Medical Science

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Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective therapy or vaccine; thus, the diagnosis and elimination of BLV-infected cattle are the most effective method to eradicate the infection. Clinical veterinarians need a simpler and more rapid method of diagnosing infection, because both nested polymerase chain reaction (PCR) and real-time PCR are labor intensive, time-consuming, and require specialized molecular biology techniques and expensive equipment. In this study, we describe a novel PCR method for amplifying the BLV provirus from whole blood, thus eliminating the need for DNA extraction. Although the sensitivity of PCR directly from whole blood (PCR-DB) samples as measured in bovine blood containing BLV-infected cell lines was lower than that of nested PCR, the PCR-DB technique showed high specificity and reproducibility. Among 225 clinical samples, 49 samples were positive by nested PCR, and 37 samples were positive by PCR-DB. There were no false positive samples; thus, PCR-DB sensitivity and specificity were 75.51% and 100%, respectively. However, the provirus loads of the samples detected by nested PCR and not PCR-DB were quite low. Moreover, PCR-DB also stably amplified the BLV provirus from tumor tissue samples. PCR-DB method exhibited good reproducibility and excellent specificity and is suitable for screening of thousands of cattle, thus serving as a viable alternative to nested PCR and real-time PCR.

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Rapid quantification of hepatitis B virus DNA by direct real-time PCR from serum without DNA extraction

Cited by 13 publications

References 13 publications

A Dry-format Field-deployable Quantitative Reverse Transcriptase-polymerase Chain Reaction Assay for Diagnosis of Dengue Infections

A Dry-format Field-deployable Quantitative Reverse Transcriptase-polymerase Chain Reaction Assay for Diagnosis of Dengue Infections

Digital PCR for direct quantification of viruses without DNA extraction

Direct polymerase chain reaction from blood and tissue samples for rapid diagnosis of bovine leukemia virus infection

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