Quantitative Detection of Gene Methylated Level of Stool Samples Based on Invader Assay Coupled with Real-time Polymerase Chain Reaction and Its Application in Non-invasive Screening of Colorectal Cancer

AbstractMethylated septin 9 (SEPT9) has been approved for non-invasive screening of colorectal cancer (CRC), but data on monitoring of CRC is sparse. Droplet digital polymerase chain reaction (ddPCR), with higher detection precision and simpler quantification than conventional PCR, has not been applied in SEPT9 detection. We explored the role of SEPT9 ddPCR for CRC detection and to measure serial SEPT9 levels in blood samples of CRC patients before and 3-month after surgery. SEPT9 methylated ratio, methylated abundance, and CEA levels were all higher in CRC patients than normal controls (all P < 0.05). The area under the curve (AUC) for methylated ratio and abundance to detect CRC was 0.707 and 0.710, respectively. There was an increasing trend for SEPT9 methylated abundance from proximal to distal cancers (P = 0.017). At 3-month after surgery, both methylated abundance and ratio decreased (P = 0.005 and 0.053, respectively), especially methylated abundance in stage III and distal cancer (both P < 0.01). We have developed a ddPCR platform for the quantitative detection of plasma SEPT9 in CRC patients. SEPT9 methylated abundance had an early post-operative decline, which may be useful in monitoring of treatment response.

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Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes

Oral Microbiology and Immunology ◽

10.1111/j.0902-0055.2004.00135.x ◽

2004 ◽

Vol 19 (3) ◽

pp. 168-176 ◽

Cited By ~ 111

Author(s):

M. Kuboniwa ◽

A. Amano ◽

K. R. Kimura ◽

S. Sekine ◽

S. Kato ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Quantitative Detection ◽

Periodontal Pathogens ◽

Chain Reaction ◽

Taqman Probes ◽

Polymerase Chain

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Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

Archives of Pathology & Laboratory Medicine ◽

10.5858/134.3.444 ◽

2010 ◽

Vol 134 (3) ◽

pp. 444-448 ◽

Cited By ~ 1

Author(s):

Zhengming Gu ◽

Jianmin Pan ◽

Matthew J. Bankowski ◽

Randall T. Hayden

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Polymerase Chain Reaction ◽

Bk Virus ◽

Clinical Samples ◽

Quantitative Detection ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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