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Multicolor combinatorial probe coding for real-time PCR.

Huang Q, Zheng L, Zhu Y, Zhang J, Wen H, Huang J, Niu J, Zhao X, Li Q - PLoS ONE (2011)

Bottom Line: MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction.This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR.MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

View Article: PubMed Central - PubMed

Affiliation: Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, Department of Biomedical Sciences and the Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, China.

ABSTRACT
The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed "Multicolor Combinatorial Probe Coding" (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2(n)-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

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Related in: MedlinePlus

Representative results of real-time PCR quantification with MCPC for S. aureus, V. cholerae, Shigella spp. and S. typhi.Purified DNA templates were 10-fold serially diluted from 100 ng to 100 fg and water was used as non-template control. The MCPC signature is given for each bacterium type. FAM: green; HEX: orange; ROX: red; and Cy5: blue.
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pone-0016033-g003: Representative results of real-time PCR quantification with MCPC for S. aureus, V. cholerae, Shigella spp. and S. typhi.Purified DNA templates were 10-fold serially diluted from 100 ng to 100 fg and water was used as non-template control. The MCPC signature is given for each bacterium type. FAM: green; HEX: orange; ROX: red; and Cy5: blue.

Mentions: Our results showed that adoption of HAND improved analytical sensitivity of the 10-plex MCPC assay to the same level of the uniplex real-time PCR (Table 1 and Figure 3), which was 100- to 1000-fold higher than the previous 8-plex PCR assay [9]. The specificity of this 10-plex MCPC system was validated using a blind test of 157 bacterial samples including the 10 foodborne pathogens and 14 other types of pathogens (Supplementary Table S1). The results showed that all 10 foodborne pathogens were correctly identified and no false-positive signal was observed when samples contained 14 non-related pathogens. The usefulness of this 10-plex MCPC assay was recognized by assembling the reagents into a test kit for routine testing in Xiamen CDC for screening of food-poisoning bacteria. Taken together, our results demonstrated that while multiplex MCPC assay was as specific as a classical probe-based real-time PCR assay; it could also be as sensitive as a uniplex PCR assay when combined with HAND system for primer design. This improvement ensured the robustness of MCPC in clinical settings.


Multicolor combinatorial probe coding for real-time PCR.

Huang Q, Zheng L, Zhu Y, Zhang J, Wen H, Huang J, Niu J, Zhao X, Li Q - PLoS ONE (2011)

Representative results of real-time PCR quantification with MCPC for S. aureus, V. cholerae, Shigella spp. and S. typhi.Purified DNA templates were 10-fold serially diluted from 100 ng to 100 fg and water was used as non-template control. The MCPC signature is given for each bacterium type. FAM: green; HEX: orange; ROX: red; and Cy5: blue.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3021529&req=5

pone-0016033-g003: Representative results of real-time PCR quantification with MCPC for S. aureus, V. cholerae, Shigella spp. and S. typhi.Purified DNA templates were 10-fold serially diluted from 100 ng to 100 fg and water was used as non-template control. The MCPC signature is given for each bacterium type. FAM: green; HEX: orange; ROX: red; and Cy5: blue.
Mentions: Our results showed that adoption of HAND improved analytical sensitivity of the 10-plex MCPC assay to the same level of the uniplex real-time PCR (Table 1 and Figure 3), which was 100- to 1000-fold higher than the previous 8-plex PCR assay [9]. The specificity of this 10-plex MCPC system was validated using a blind test of 157 bacterial samples including the 10 foodborne pathogens and 14 other types of pathogens (Supplementary Table S1). The results showed that all 10 foodborne pathogens were correctly identified and no false-positive signal was observed when samples contained 14 non-related pathogens. The usefulness of this 10-plex MCPC assay was recognized by assembling the reagents into a test kit for routine testing in Xiamen CDC for screening of food-poisoning bacteria. Taken together, our results demonstrated that while multiplex MCPC assay was as specific as a classical probe-based real-time PCR assay; it could also be as sensitive as a uniplex PCR assay when combined with HAND system for primer design. This improvement ensured the robustness of MCPC in clinical settings.

Bottom Line: MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction.This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR.MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

View Article: PubMed Central - PubMed

Affiliation: Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, Department of Biomedical Sciences and the Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, China.

ABSTRACT
The target volume of multiplex real-time PCR assays is limited by the number of fluorescent dyes available and the number of fluorescence acquisition channels present in the PCR instrument. We hereby explored a probe labeling strategy that significantly increased the target volume of real-time PCR detection in one reaction. The labeling paradigm, termed "Multicolor Combinatorial Probe Coding" (MCPC), uses a limited number (n) of differently colored fluorophores in various combinations to label each probe, enabling one of 2(n)-1 genetic targets to be detected in one reaction. The proof-of-principle of MCPC was validated by identification of one of each possible 15 human papillomavirus types, which is the maximum target number theoretically detectable by MCPC with a 4-color channel instrument, in one reaction. MCPC was then improved from a one-primer-pair setting to a multiple-primer-pair format through Homo-Tag Assisted Non-Dimer (HAND) system to allow multiple primer pairs to be included in one reaction. This improvement was demonstrated via identification of one of the possible 10 foodborne pathogen candidates with 10 pairs of primers included in one reaction, which had limit of detection equivalent to the uniplex PCR. MCPC was further explored in detecting combined genotypes of five β-globin gene mutations where multiple targets were co-amplified. MCPC strategy could expand the scope of real-time PCR assays in applications which are unachievable by current labeling strategy.

Show MeSH
Related in: MedlinePlus