Norovirus Real Time RT-PCR Detection Technology Transition to the Joint Biological Identification and Diagnosis System (JBAIDS)

Abstract Citrus bark cracking viroid (CBCVd), formerly known as pathogen in the genus Citrus and first detected in Slovenian hops in 2014, threatens hop production as it leads to important economic losses. Reduction in yield and quality and even death of the infected plants within a few years are typical observations due to CBCVd infections of hops. The viroid is easily transmitted and spreads rapidly. As it cannot be controlled by plant protection measures, avoiding its introduction into hop gardens and eradicating first centres of infection are of utmost importance. An indispensable prerequisite is a reliable detection method suitable for large-scale routine testing. In this study, the development of primers and probe for real-time RT-PCR for sensitive CBCVd detection is described. To exclude “false negative” results, a nad5 mRNA-based internal positive control was included. To our knowledge, this is the first time such a duplex real-time RT-PCR detection method for CBCVd at least in hops is described. In addition, first method validation data are presented.

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The use and correlation with flow cytometry of fluorescent molecular beacons in real time PCR of IgH gene rearrangements for evaluation of minimal residual disease in multiple myeloma

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.17552 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 17552-17552

Author(s):

O. Kara ◽

A. Yigin ◽

B. Sahin ◽

S. Paydas

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Real Time ◽

Minimal Residual Disease ◽

Real Time Pcr ◽

Residual Disease ◽

Pcr Detection ◽

Molecular Beacons ◽

Rt Pcr ◽

Minimal Residual

17552 Background: At present, the prognostic value of the amount of residual tumor cells in PB, BM or stem cell harvests and its changes over time is stil not clear. Also the advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by using flow cytometry. The aim of the present study was to evaluate a real time PCR test for the IgH gene using alellespecific molecular beacons as fluorescence probes to quantify residual disease and also correlate flow cytometric detection of plasma cells in MM patients during followup after treatment with high dose of chemotherapy or standart chemotherapy. Methods: After clinical diagnosis of 17 MM patients, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify its clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. We have also examined the co-expression of CD19, CD38, CD45, CD56, and CD138 molecules in cells of bone marrow aspirates in patients with multiple myeloma by flow cytometry. Results: The active disease had been accepted of whom plasma cell infiltration ratio was over 10% in bone marrow and also of whom labeled by CD38 and CD138 by FCM. The detection of the MRD was positive in 13 patients by RT-PCR, respectively. The infiltration ratio was correlated with CD138 expression (p = 0.009) and RT-PCR detection of plasma cells (p = 0.006) and also significant correlation had been found between RT-PCR detection and CD138 expression respectively. No any correlaton was found between other surface antigens (CD38, CD45, CD56). Conclusion: Our results indicated that real time PCR with specific molecular beacons provides a feasible, accurate and reproducible method for the determination of minimal residual disease in MM. By FCM only CD138 expression may have been used as disease marker in addition of the RT-PCR detection. No significant financial relationships to disclose.

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