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PET-PCR method for the molecular detection of malaria parasites in a national malaria surveillance study in Haiti, 2011.

Lucchi NW, Karell MA, Journel I, Rogier E, Goldman I, Ljolje D, Huber C, Mace KE, Jean SE, Akom EE, Oscar R, Buteau J, Boncy J, Barnwell JW, Udhayakumar V - Malar. J. (2014)

Bottom Line: A total of 12 out of the 2,989 samples screened (0.4%) were found to be positive by PET-PCR (mean CT value of 35.7).These same samples were also found to be positive by the nested and TaqMan-based methods.While the nested PCR was found to be slightly more sensitive than the PET-PCR, it is not ideal for high throughput screening of samples.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, Center for Global Health, Division of Parasitic Diseases and Malaria, Malaria Branch, Atlanta, GA, USA. NLucchi@cdc.gov.

ABSTRACT

Background: Recently, a real-time PCR assay known as photo-induced electron transfer (PET)-PCR which relies on self-quenching primers for the detection of Plasmodium spp. and Plasmodium falciparum was described. PET-PCR assay was found to be robust, and easier to use when compared to currently available real-time PCR methods. The potential of PET-PCR for molecular detection of malaria parasites in a nationwide malaria community survey in Haiti was investigated.

Methods: DNA from the dried blood spots was extracted using QIAGEN methodology. All 2,989 samples were screened using the PET-PCR assay in duplicate. Samples with a cycle threshold (CT) of 40 or less were scored as positive. A subset of the total samples (534) was retested using a nested PCR assay for confirmation. In addition, these same samples were also tested using a TaqMan-based real-time PCR assay.

Results: A total of 12 out of the 2,989 samples screened (0.4%) were found to be positive by PET-PCR (mean CT value of 35.7). These same samples were also found to be positive by the nested and TaqMan-based methods. The nested PCR detected an additional positive sample in a subset of 534 samples that was not detected by either PET-PCR or TaqMan-based PCR method.

Conclusion: While the nested PCR was found to be slightly more sensitive than the PET-PCR, it is not ideal for high throughput screening of samples. Given the ease of use and lower cost than the nested PCR, the PET-PCR provides an alternative assay for the rapid screening of a large number of samples in laboratory settings.

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Related in: MedlinePlus

Sample processing.
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Fig1: Sample processing.

Mentions: The DBS were transferred to the CDC Atlanta, GA, USA, for molecular diagnosis. A complete database of all received DBS was established upon receipt of the samples. Each DBS was carefully examined for signs of potential contamination and to make sure there was sufficient volume of blood before DNA was extracted. Out of the 3,041 DBS received, 52 were excluded (Figure 1). DNA was successfully extracted from the remaining 2,989 samples using the QIAamp DNA Mini Kits (QIAGEN, Valencia, CA, USA) as described by the manufacturer. Briefly, three 3-mm punches of the DBS were punched out and placed into a 1.5-mL tube for processing, according to instructions. The DNA was eluted in 150 μL of elution buffer, aliquoted and stored at -20°C until use.Figure 1


PET-PCR method for the molecular detection of malaria parasites in a national malaria surveillance study in Haiti, 2011.

Lucchi NW, Karell MA, Journel I, Rogier E, Goldman I, Ljolje D, Huber C, Mace KE, Jean SE, Akom EE, Oscar R, Buteau J, Boncy J, Barnwell JW, Udhayakumar V - Malar. J. (2014)

Sample processing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289323&req=5

Fig1: Sample processing.
Mentions: The DBS were transferred to the CDC Atlanta, GA, USA, for molecular diagnosis. A complete database of all received DBS was established upon receipt of the samples. Each DBS was carefully examined for signs of potential contamination and to make sure there was sufficient volume of blood before DNA was extracted. Out of the 3,041 DBS received, 52 were excluded (Figure 1). DNA was successfully extracted from the remaining 2,989 samples using the QIAamp DNA Mini Kits (QIAGEN, Valencia, CA, USA) as described by the manufacturer. Briefly, three 3-mm punches of the DBS were punched out and placed into a 1.5-mL tube for processing, according to instructions. The DNA was eluted in 150 μL of elution buffer, aliquoted and stored at -20°C until use.Figure 1

Bottom Line: A total of 12 out of the 2,989 samples screened (0.4%) were found to be positive by PET-PCR (mean CT value of 35.7).These same samples were also found to be positive by the nested and TaqMan-based methods.While the nested PCR was found to be slightly more sensitive than the PET-PCR, it is not ideal for high throughput screening of samples.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, Center for Global Health, Division of Parasitic Diseases and Malaria, Malaria Branch, Atlanta, GA, USA. NLucchi@cdc.gov.

ABSTRACT

Background: Recently, a real-time PCR assay known as photo-induced electron transfer (PET)-PCR which relies on self-quenching primers for the detection of Plasmodium spp. and Plasmodium falciparum was described. PET-PCR assay was found to be robust, and easier to use when compared to currently available real-time PCR methods. The potential of PET-PCR for molecular detection of malaria parasites in a nationwide malaria community survey in Haiti was investigated.

Methods: DNA from the dried blood spots was extracted using QIAGEN methodology. All 2,989 samples were screened using the PET-PCR assay in duplicate. Samples with a cycle threshold (CT) of 40 or less were scored as positive. A subset of the total samples (534) was retested using a nested PCR assay for confirmation. In addition, these same samples were also tested using a TaqMan-based real-time PCR assay.

Results: A total of 12 out of the 2,989 samples screened (0.4%) were found to be positive by PET-PCR (mean CT value of 35.7). These same samples were also found to be positive by the nested and TaqMan-based methods. The nested PCR detected an additional positive sample in a subset of 534 samples that was not detected by either PET-PCR or TaqMan-based PCR method.

Conclusion: While the nested PCR was found to be slightly more sensitive than the PET-PCR, it is not ideal for high throughput screening of samples. Given the ease of use and lower cost than the nested PCR, the PET-PCR provides an alternative assay for the rapid screening of a large number of samples in laboratory settings.

Show MeSH
Related in: MedlinePlus