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Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China.

Wu L, Yin X, Zheng L, Zou J, Jin P, Hu Y, Kudinha T, Kong F, Chen X, Wang Q - PLoS ONE (2015)

Bottom Line: However, only certain serotypes are more likely to cause pneumococcal diseases.Thus serotype surveillance is important for vaccine formula design as well as in post-vaccine serotype shift monitor.We designed serotype/serogroup-specific MLPA probes and fluorescent detection probes to discriminate the different serotypes/serogroups in one molecular reaction.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Medical Centre, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China; Clinical Laboratory, Bao'an Maternity and Child Health Hospital, Shenzhen, Guangdong Province, China.

ABSTRACT

Background: Streptococcus pneumoniae has more than 95 distinct serotypes described to date. However, only certain serotypes are more likely to cause pneumococcal diseases. Thus serotype surveillance is important for vaccine formula design as well as in post-vaccine serotype shift monitor. The goal of this study was to develop a practical screening assay for ten Shenzhen China common pneumococcal serotypes/serogroups in one molecular reaction.

Methods: A molecular assay, based on multiplex ligation-dependent probe amplification (MLPA) and melting curve (MC) analysis, was developed in an integrated approach (MLPA-MC) for the detection of ten capsular serotypes/serogroups 4, 6 (6A/6B/6C/6D), 9V/9A, 14, 15F/15A, 15B/15C, 18 (18F/18A/18B/18C), 19F, 19A and 23F. We designed serotype/serogroup-specific MLPA probes and fluorescent detection probes to discriminate the different serotypes/serogroups in one molecular reaction. The three steps of MLPA-MC assay are continuous reactions in one well detected by LightCycler 480. A total of 210 S. pneumoniae isolates from our local Maternity and Child Health Hospital were randomly chosen to evaluate the assay against published multiplex PCR assays.

Results: Our results showed that 198 (94.3%) of S. pneumoniae isolates were type-able by our assays and the results were in complete concordance with the published multiplex PCRs. Using the MLPA-MC assay, 96 S. pneumoniae isolates could be typed within 3 hours with limited hands-on time. This serotype/serogroup-screening assay can be easily modified or extended by modification of the serotype/serogroup-specific MLPA probes combinations according to the needs of different laboratories.

Conclusions: We recommend use of this assay as a starting point for screening serotype/serogroup frequencies. There is a need for this assay to be combined with other molecular typing assays, like published serotype specific PCRs, or even the Quellung reaction for serotype confirmation.

No MeSH data available.


Related in: MedlinePlus

Melting curve analysis of one negative control isolate.(A) Melting peak of the internal control lytA (ROX dye). (B) Melting peak of the internal control cpsA (Cy5 dye).
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pone.0130664.g003: Melting curve analysis of one negative control isolate.(A) Melting peak of the internal control lytA (ROX dye). (B) Melting peak of the internal control cpsA (Cy5 dye).

Mentions: Sensitivity of MLPA-MC assay was tested on serial dilutions of cloned DNA standards with a minimal threshold for detection set at 1000 copies for each probe in the panel. Serotype/serogroup-specificities of the specific probes were accessed by testing the control isolates. When the target control isolates were tested by MLPA-MC assay, the results showed three melting peaks, the serotype/serogroup specific peak, the cpsA, and lytA peaks, as shown in Fig 2. For the other serotypes/serogroups (other than the 10 targeted serotypes/serogroups) isolates that were used as negative or specificity controls, only cpsA and lytA peaks were obtained, as in Fig 3. The actual melting peak of each pair of probes fluctuated in the range of ±0~2 centigrade (°C) to the expected Tm, which was acceptable, as shown in Fig 4. No cross-reactivity among the ten target probes was observed when they were tested against the control set. Of the ten probes, five were specific for the target serotypes of 4, 14, 19F, 19A and 23F. For USA CDC specific mPCRs, the PCR primers can’t discriminate between serotypes 9V and 9A, 15B and 15C, 15F and 15A, four serotypes of serogroup 18 (18F/18A/18B/18C) and four serotypes of serogroup 6 (6A/6B/6C/6D), respectively. In this study, serotype 9V/9A probe reacted with both serotypes 9V and 9A; serotype 15F/15A probe reacted with both serotypes 15F and 15A; serotype 15B/15C probe reacted with both serotypes 15B and 15C; serogroup 18 probe can react with all 4 serotypes of 18F/18A/18B/18C; serogroup 6 probe can react with all 4 serotypes of 6A/6B/6C/6D. Reproducibility of our results depended on the purity of DNA samples and the thoroughness of mixing during the reactions.


Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China.

Wu L, Yin X, Zheng L, Zou J, Jin P, Hu Y, Kudinha T, Kong F, Chen X, Wang Q - PLoS ONE (2015)

Melting curve analysis of one negative control isolate.(A) Melting peak of the internal control lytA (ROX dye). (B) Melting peak of the internal control cpsA (Cy5 dye).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495002&req=5

pone.0130664.g003: Melting curve analysis of one negative control isolate.(A) Melting peak of the internal control lytA (ROX dye). (B) Melting peak of the internal control cpsA (Cy5 dye).
Mentions: Sensitivity of MLPA-MC assay was tested on serial dilutions of cloned DNA standards with a minimal threshold for detection set at 1000 copies for each probe in the panel. Serotype/serogroup-specificities of the specific probes were accessed by testing the control isolates. When the target control isolates were tested by MLPA-MC assay, the results showed three melting peaks, the serotype/serogroup specific peak, the cpsA, and lytA peaks, as shown in Fig 2. For the other serotypes/serogroups (other than the 10 targeted serotypes/serogroups) isolates that were used as negative or specificity controls, only cpsA and lytA peaks were obtained, as in Fig 3. The actual melting peak of each pair of probes fluctuated in the range of ±0~2 centigrade (°C) to the expected Tm, which was acceptable, as shown in Fig 4. No cross-reactivity among the ten target probes was observed when they were tested against the control set. Of the ten probes, five were specific for the target serotypes of 4, 14, 19F, 19A and 23F. For USA CDC specific mPCRs, the PCR primers can’t discriminate between serotypes 9V and 9A, 15B and 15C, 15F and 15A, four serotypes of serogroup 18 (18F/18A/18B/18C) and four serotypes of serogroup 6 (6A/6B/6C/6D), respectively. In this study, serotype 9V/9A probe reacted with both serotypes 9V and 9A; serotype 15F/15A probe reacted with both serotypes 15F and 15A; serotype 15B/15C probe reacted with both serotypes 15B and 15C; serogroup 18 probe can react with all 4 serotypes of 18F/18A/18B/18C; serogroup 6 probe can react with all 4 serotypes of 6A/6B/6C/6D. Reproducibility of our results depended on the purity of DNA samples and the thoroughness of mixing during the reactions.

Bottom Line: However, only certain serotypes are more likely to cause pneumococcal diseases.Thus serotype surveillance is important for vaccine formula design as well as in post-vaccine serotype shift monitor.We designed serotype/serogroup-specific MLPA probes and fluorescent detection probes to discriminate the different serotypes/serogroups in one molecular reaction.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Medical Centre, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China; Clinical Laboratory, Bao'an Maternity and Child Health Hospital, Shenzhen, Guangdong Province, China.

ABSTRACT

Background: Streptococcus pneumoniae has more than 95 distinct serotypes described to date. However, only certain serotypes are more likely to cause pneumococcal diseases. Thus serotype surveillance is important for vaccine formula design as well as in post-vaccine serotype shift monitor. The goal of this study was to develop a practical screening assay for ten Shenzhen China common pneumococcal serotypes/serogroups in one molecular reaction.

Methods: A molecular assay, based on multiplex ligation-dependent probe amplification (MLPA) and melting curve (MC) analysis, was developed in an integrated approach (MLPA-MC) for the detection of ten capsular serotypes/serogroups 4, 6 (6A/6B/6C/6D), 9V/9A, 14, 15F/15A, 15B/15C, 18 (18F/18A/18B/18C), 19F, 19A and 23F. We designed serotype/serogroup-specific MLPA probes and fluorescent detection probes to discriminate the different serotypes/serogroups in one molecular reaction. The three steps of MLPA-MC assay are continuous reactions in one well detected by LightCycler 480. A total of 210 S. pneumoniae isolates from our local Maternity and Child Health Hospital were randomly chosen to evaluate the assay against published multiplex PCR assays.

Results: Our results showed that 198 (94.3%) of S. pneumoniae isolates were type-able by our assays and the results were in complete concordance with the published multiplex PCRs. Using the MLPA-MC assay, 96 S. pneumoniae isolates could be typed within 3 hours with limited hands-on time. This serotype/serogroup-screening assay can be easily modified or extended by modification of the serotype/serogroup-specific MLPA probes combinations according to the needs of different laboratories.

Conclusions: We recommend use of this assay as a starting point for screening serotype/serogroup frequencies. There is a need for this assay to be combined with other molecular typing assays, like published serotype specific PCRs, or even the Quellung reaction for serotype confirmation.

No MeSH data available.


Related in: MedlinePlus