Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction

Abstract Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches.Methods: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method developed will be useful in disease association studies involving large cohorts.

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A NEW REAL-TIME RT-PCR. METHOD FOR THE DETECTION OF PSA POSITIVE CELLS IN BLOOD OF PROSTATE CANCER PATIENTS

European Urology Supplements ◽

10.1016/s1569-9056(06)61257-8 ◽

2006 ◽

Vol 5 (14) ◽

pp. 788

Author(s):

G. Barbero ◽

P. Destefanis ◽

S. Procida ◽

C. Fiori ◽

C. Ceruti ◽

...

Keyword(s):

Prostate Cancer ◽

Real Time ◽

Cancer Patients ◽

Rt Pcr ◽

Pcr Method

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Evaluation of a 384–Well Format for High-Throughput Real-Time Reverse Transcription Polymerase Chain Reaction for Avian Influenza Testing

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870902100513 ◽

2009 ◽

Vol 21 (5) ◽

pp. 679-683 ◽

Cited By ~ 4

Author(s):

Pamela J. Ferro ◽

Jason Osterstock ◽

Bo Norby ◽

Geoffrey T. Fosgate ◽

Blanca Lupiani

Keyword(s):

Polymerase Chain Reaction ◽

Avian Influenza ◽

Real Time ◽

High Throughput ◽

Reverse Transcription ◽

Virus Isolation ◽

Rt Pcr ◽

Chain Reaction ◽

Response Planning ◽

Polymerase Chain

As concerns over the global spread of highly pathogenic avian influenza H5N1 have heightened, more countries are faced with increased surveillance efforts and incident response planning for handling a potential outbreak. The incorporation of molecular techniques in most diagnostic laboratories has enabled fast and efficient testing of many agents of concern, including avian influenza. However, the need for high-throughput testing remains. In this study, the use of a 384–well format for high-throughput real-time reverse transcription polymerase chain reaction (real-time RT-PCR) testing for avian influenza is described. The analytical sensitivity of a real-time RT-PCR assay for avian influenza virus matrix gene with the use of both 96– and 384–well assay formats and serial dilutions of transcribed control RNA were comparable, resulting in similar limits of detection. Of 28 hunter-collected cloacal swabs that were positive by virus isolation, 26 (92.9%) and 27 (96.4%) were positive in the 96– and 384–well assays, respectively; of the 340 hunter-collected swabs that were negative by virus isolation, 45 (13.2%) and 23 (6.8%) were positive in the 96– and 384–well assays, respectively. The data presented herein supports the utility of the 384–well format in the event of an avian influenza outbreak for high-throughput real-time RT-PCR testing.

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