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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgments
 ~  References
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 33  |  Issue : 4  |  Page : 516-523
 

Optimization and head-to-head comparison of MISSR-PCR, ERIC-PCR, RAPD and 16S rRNA evolutionary clock for the genotyping of Vibrio cholerae isolated in China


1 Key Laboratory of Diarrhea Disease Detection Zhuhai International Travel Healthcare Center, Zhuhai Entry-Exit Inspection and Quarantine Bureau, Zhuhai, China
2 Department of Biostatistics, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong, China

Date of Submission19-Mar-2014
Date of Acceptance22-Apr-2015
Date of Web Publication16-Oct-2015

Correspondence Address:
Z Yang
Key Laboratory of Diarrhea Disease Detection Zhuhai International Travel Healthcare Center, Zhuhai Entry-Exit Inspection and Quarantine Bureau, Zhuhai
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.167321

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 ~ Abstract 

Purpose: To establish a new genotyping method for Vibrio cholerae and compare it with other methods. Materials and Methods: In the current study, a modified inter simple sequence repeat-polymerase chain reaction (MISSR-PCR) system was developed via several rounds of optimisation. Comparison study was then conducted between MISSR-PCR and three other methods, including enterobacterial repetitive intergenic consensus sequences-based PCR (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) and 16S rRNA evolutionary clock, for the detection and genetic tracing of Vibrio cholerae isolated from seafood in China. Result: The results indicated that the MISSR-PCR system could generate the highest polymorphic fingerprinting map in a single round PCR and showed the best discriminatory ability for Vibrio cholerae genotyping by clearly separating toxigenic/nontoxigenic strains, local/foreign strains, and O1/O139/non-O1/non-O139 serogroup strains, comparing to ERIC-PCR, RAPD and 16S rRNA evolutionary clock. Moreover, the MISSR-PCR is superior to previously described traditional simple sequence repeat based PCR method on genotyping by more clearly separating different clusters. Conclusion: To the best of our knowledge, this is the first head-to-head comparison of four detection and genotyping methods for Vibrio cholerae The MISSR-PCR system established here could serve as a simple, quick, reliable and cost-effective tool for the genotyping and epidemiological study.


Keywords: Detection, food safety, genotyping, modified inter simple sequence repeat-polymerase chain reaction ,, Vibrio cholera


How to cite this article:
Mo Q H, Wang H B, Tan H, An S L, Feng Z L, Wang Q, Lin J C, Yang Z. Optimization and head-to-head comparison of MISSR-PCR, ERIC-PCR, RAPD and 16S rRNA evolutionary clock for the genotyping of Vibrio cholerae isolated in China. Indian J Med Microbiol 2015;33:516-23

How to cite this URL:
Mo Q H, Wang H B, Tan H, An S L, Feng Z L, Wang Q, Lin J C, Yang Z. Optimization and head-to-head comparison of MISSR-PCR, ERIC-PCR, RAPD and 16S rRNA evolutionary clock for the genotyping of Vibrio cholerae isolated in China. Indian J Med Microbiol [serial online] 2015 [cited 2019 Dec 12];33:516-23. Available from: http://www.ijmm.org/text.asp?2015/33/4/516/167321



 ~ Introduction Top


To ensure food safety, it is of great importance to monitor and detect pathogens hidden in food. Vibrio cholerae is among such kind of pathogens and is the causative agent for cholera in human beings. Till now, cholera remains a global threat to public health, especially for people living in developing countries. From 2004, there is a steady increase in annual reported cholera cases, with a peak in 2011, during when a total of 58 countries from all of the five continents reporting a cumulative sum of 589,854 cases, including 7,816 deaths in this single year.[1] The real number is thought to be much bigger than this statistics, due to the poor surveillance and diagnosis of cholera. According to an estimation, the actual number of cholera cases is as high as 2.8 million (uncertainty range: 1.2–4.3 million), with about 91,000 deaths (uncertainty range: 28,000–142,000) each year.[2]

Vibrio cholerae is a gram-negative bacterium which is naturally present in water and possibly in cultured fish or seafood.[3] By serological study, more than 200 O-antigen serogroups have been defined, in which serogroup O1 (including Classical biotypes and El Tor biotypes) and O139 are usually toxigenic and can cause epidemic [4] while other Vibrio cholerae serogroups are nontoxigenic and not associated with epidemics, which are collectively referred to as nonO1/non O139 serogroups.[5] However, due to the horizontal gene transfer,[6] there are several reports on the loss of cholera toxin producing ability of some O1 serogroup strains,[7] as well as some non-O1/non-O139 serogroup strains serving as the causative agents of sporadic cases of cholera-like disease,[8] indicating the complexity on differentiating toxigenic/nontoxigenic strains and O1/O139/non-O1/non-O139 serogroup strains. In addition, for epidemiological studies, it is also necessary to differentiate local strains from foreign strains.

Molecular methods for detection and genotyping are accurate approaches, which are of great importance for the diagnosis and epidemiological study. Traditional molecular methods, such as ribotyping [9] and pulsed-field gel electrophoresis (PFGE),[10] are time-consuming and expensive, which limit their routine use in clinical laboratories or technical confined laboratories. In contrast, newly developed PCR-based genotyping methods, including inter simple sequence repeat-polymerase chain reaction (ISSR-PCR),[11] enterobacterial repetitive intergenic consensus sequences-based PCR (ERIC-PCR),[12] randomly amplified polymorphic DNA (RAPD)[13] and 16S rRNA evolutionary clock [14] are easier and faster, which are widely used for prokaryotic genotyping.

However, till now there is only one research group from India who used ISSR-PCR system for Vibrio cholerae genotyping.[11] Though toxigenic/non-toxigenic strains could be separated clearly by this traditional ISSR-PCR system, they could not generate ideal fingerprinting profiles to distinguish O1 serogroup strains from O139 serogroup strains. Therefore, one aim of the current study is to optimise and establish a highly discriminative modified ISSR-PCR (MISSR-PCR) system for detection and genotyping of Vibrio cholerae specifically. In addition, as no previous study compared those genotyping methods simultaneously, a head-to-head comparison among these methods was conducted. Direct PCR on the virulence gene ctxA in these strains was used to confirm the cluster analysis results.


 ~ Materials and Methods Top


Bacterial strains

A total of 72 Vibrio cholerae strains isolated from seafood were selected, which were the maximum quantity of the strains obtained during the past several years. These strains included fifty O1 serogroup strains (A-1 to A-13, A-15 to A-33, A-36 to A-44, A-56, A-67 to A-71, A-73 to A-75), nine O139 serogroup strains (B-57 to B-65) and thirteen non-O1/non-O139 serogroup strains (N-14, N-34, N-35, N-45 to N-54). O139 serogroup reference strain MO45 was also included for comparison.[15]

PCR fingerprinting

Highly pure genomic DNA was prepared using Bacteria Genomic DNAout kit (Tiandz, Inc, Beijing, China) according to the manufacturer's instruction. Primers are listed in [Table 1],[Table 2],[Table 3]. PCR amplifications were conducted using the following optimised conditions: (1) MISSR-PCR: the 25 µl reaction system contained 2.5 μl 10X PCR buffer, 1.2 μM optimised primer, 2.0 mM MgCl2, 0.3 mM of each dNTP, 0.5 U of Taq polymerase (Takara, Dalian, China), DNA 3 μl (5 ng/ul), and the cycling programme was 95°C 3 min, followed by 30 cycles of 94°C 40 sec, 45°C 1 min, 72°C 2 min, and a final 72°C 10 min; (2) ERIC-PCR: The 25 μl reaction system contained 2.5 μl 10X PCR buffer, 1.2 μM each primer, 1.7 mM MgCl2, 0.38 mM of each dNTP, 0.5U of Taq polymerase, DNA 2 μl, and the cycling programme was 95°C 5 min, followed by 30 cycles of 94°C 30 sec, 50°C 30 sec, 52°C 30 sec, 72°C 1 min, and a final 72°C 10 min; (3) RAPD: The 20 µl reaction system contained 2 μl 10X PCR buffer, 1 μM random primer, 2.5 mM MgCl2, 0.25 mM of each dNTP, 0.5 U of Taq polymerase, DNA 2 μl, and the cycling program was 95°C 3 min, followed by 30 cycles of 94°C 40 sec, 36°C 1 min, 72°C 2 min (with a 0.3°C/sec temperature increase rate from 36–72°C), and a final 72°C 10 min. PCR products were analysed on 2% agarose gel and documented using the ImageLab 2.0 system (Bio-Rad Laboratories, CA, USA). The amplifications were repeated three times to evaluate reproducibility.
Table 1: Primer list for MISSR-PCR optimization

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Table 2: Primer list for RAPD optimization

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Table 3: Primer selected/used

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16S rRNA amplification and sequencing

The 16S rRNA gene was amplified by regular PCR using the specific primers in [Table 3]. The 1533 bp 16S rRNA fragment was checked on 1% agarose gel and then sent out for sequencing using the same pair of primers directly.

Cluster analysis and construction of phylogenetic tree

Specific and reproducible MISSR-PCR, ERIC-PCR and RAPD amplicons were selected and scored for the presence as 1 or absence as 0. The size of the amplicons was calculated using the ImageLab 2.0 analysis software. Cluster analysis (average linkage between groups) was performed using SPSS 17.0 software package (SPSS Science, IL, USA). For 16S rRNA gene, nucleotide sequences were aligned with the reference strain CP003069 using the Clustalx 1.81 programme.[21] The genetic distance was calculated using Kimura's two-parameter model.[22] Phylogenetic tree was generated using the neighbor-joining method which was implemented in the Mega 4.0 programme. The tree was printed using the TreeView program.

Detection of virulence genes

To further confirm our results, Vibrio cholerae specific primers targeting the outer membrane protein (ompW) and virulence gene cholera toxin A subunit (ctxA) were used.[19],[20] Regular PCR was conducted and the PCR products were analysed on 2% agarose gel.


 ~ Results Top


The optimization and establishment of MISSR-PCR system

In order to design appropriate MISSR-PCR primers, the prokaryotic microsatellite database, MICdb (http://www.cdfd.org.in/micas)[23] was used to analyse the frequency of SSR repeats in the genome of Vibrio cholerae. Di-nucleotides motifs (complementary repeat motifs were grouped into one class, such as GC/CG) repeating a minimum of four times were considered. When reference strain CP001235 was analyzed, GA/CT, AG/TC and GC/CG showed higher abundance compared to AT/TA (data not shown). Based on this analysis, eight anchored MISSR primers [Table 1], No. 1-8] were designed. Preliminary data showed that smear or limited bands were generated by those primers. Only primer MISSR_cGA7 could generate clear bands but with limited polymorphism [Figure 1], left panel]. Therefore, multi-runs of optimisation for the MISSR-PCR system was conducted. Firstly the length of the primers was decreased in order to reduce the amplification specificity and increase the polymorphism. Therefore, primers with GA six-repeats, GA five-repeats, GA four-repeats, AG seven-repeats, AG six-repeats, AG five-repeats and AG four-repeats [Table 1], No. 9-16] were tried. Secondly different concentration of Mg 2+ (5.0 mM, 4.0 mM, 3.0 mM, 2.0 mM, 1.0 mM and 0.5 mM) was used. Then the concentration of dNTPs (1.0 mM, 0.5 mM, 0.3 mM, 0.2 mM, 0.1 mM and 0.05 mM) was optimised when the concentration of Mg 2+ was fixed. Similar approach was also applied to the concentration of primers (3.0 μM, 2.0 μM, 1.5 μM, 1.2 μM, 0.6 μM and 0.3 μM). By comparing the fingerprinting map generated (clarity, polymorphism) and the stability of the each system, finally the system was fixed to use 1.2 μM MISSR_GA5 as the primer, with 2.0 mM MgCl2 and 0.3 mM of dNTPs. Detail information on the condition of the MISSR-PCR system was described in the material and methods.{Table 1}
Figure 1: Fingerprinting maps of the Vibrio cholerae strains produced by MISSR-PCR. Left panel, 22mer MISSR_cGA7 primer, before optimisation; right panel, 12mer MISSR_GA5 primer, after optimization. No. 55 is the reference strain MO45. Arrow indicates the specific marker for toxigenic strains. M, 100 bp ladder

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MISSR-PCR fingerprinting

The 12mer primer MISSR_GA5 generated 28 scorable bands, varying from 150–3000 bp [Figure 1], right panel]. This fingerprinting map was clear and highly polymorphic, which was suitable for genotyping analysis. Since many of the Vibrio cholerae strains showed exactly the same pattern on fingerprinting map [Figure 1], only representative strains were chose for analysis. The dendrogram generated based on the cluster analysis on 16 representing strains (including eleven O1 serogroup strains, two O139 serogroup strains and three non-O1/non-O139 serogroup strains) showed high discriminative ability between toxigenic (A-68, A-69, B-58 and MO45) and nontoxigenic trains [Figure 2]a. In the toxigenic cluster, two local O1 serogroup strains (A-68 and A-69) clustered tightly and then formed a small subcluster with another local O139 serogroup stains (B-58). The distance existing between this subcluster and the India-origin O139 serogroup strain MO45 indicated that the MISSR-PCR system could distinguish not only toxigenic strains and nontoxigenic trains but also local strains and foreign strains. The rest nontoxigenic strains could be divided into two clear subclusters, O1 serogroup strains and non-O1/non-O139 serogroup strains, with only one exception (A-70). Moreover, a toxigenic strain specific marker [a strong 350 bp band, [Figure 1], right panel] was identified by detailed analysis of the fingerprinting data, which would assist the quick discrimination of toxigenic and nontoxigenic strains. Screening for the presence of virulence gene ctxA in these strains confirmed the cluster analysis results [Figure 2]b.
Figure 2: Genotyping results of MISSR-PCR. (a) The MISSR-PCR profile-derived dendrogram illustrating the clustering of the 16 representative Vibrio cholerae strains. *O1 serogroup strain clustered with the non-O1/non-O139 serogroup strains due to genome sequence variation. (b) PCR amplification of the outer membrane protein gene ompW and virulence gene ctxA

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ERIC-PCR fingerprinting

After optimization the ERIC-PCR system, a clear fingerprinting map with 25 scorable bands, varying from 150–2200 bp [Figure 3]a was obtained. The dendrogram [Figure 3]b based on the same panel of 16 representative isolates showed that the nontoxigenic O1 serogroup and toxigenic group were independently clustered while local and foreign strains also located apart from each other. However, in the toxigenic group, the O1 and O139 serogroup strains clustered tightly which could not be separated. In addition, there was one O1 serogroup strain A-7 and one non-O1/non-O139 serogroup strain N-45 located outside of their main clusters, respectively.
Figure 3: Genotyping results of ERIC-PCR. (a) Fingerprinting maps produced by ERIC-PCR. (b) The ERIC-PCR profile-derived dendrogram illustrating the clustering of the 16 Vibrio cholerae strains

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RAPD genotyping

Forty random primers [Table 2], kits A and D, Dingguo, Beijing, China] were initially used to run PCR and the best primer was selected based on the number, quality and polymorphism of the amplified products. Finally primer A09 was chose and RAPD fingerprinting map generated from it was applied for analysis. A09 could generate 25 clear bands, varying from 180–3000 bp [Figure 4]a. Cluster analysis [Figure 4]b based on the 16 isolates showed that it could distinguish toxigenic strain and nontoxigenic strains. In the toxigenic cluster, as described in MISSR-PCR, O1 serogroup strains and O139 serogroup strains, local strains and foreign strains could be separated clearly. Moreover, primer A09 generated a faint 320 bp band which may serve as a marker for toxigenic strains [Figure 4]a. However, in nontoxigenic cluster, the pattern was not as clear as MISSR-PCR. First, the nontoxigenic non-O1/non-O139 strain N-45 located far away from the main cluster. Second, the other two non-O1/non-O139 serogroup strains N-34 and N-35 located in the middle of the O1 serogroup strains, which separated the O1 serogroup strains into two independent subclusters [Figure 4]b.{Table 2}
Figure 4: Genotyping results of RAPD. (a) Fingerprinting maps produced by RAPD. Arrow indicates the specific marker for toxigenic strains. (b) The RAPD profile-derived dendrogram illustrating the clustering of the 16 Vibrio cholerae strains

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16S rRNA evolutionary clock

After PCR and direct sequencing, 16S rRNA gene sequences from the 16 selected strains were successfully obtained. The similarity among these sequences was 99.88% with only few point mutations. Phylogenetic tree indicated that there were two major clusters [Figure 5]. However, different serogroups could not be distinguished. For example, non-O1/non-O139 serogroup strain N-35 located in the O1 serogroup cluster while the two O139 serogroup strains B-58 and MO45 were in another O1 serogroup cluster. Moreover, the local and foreign isolates could not be separated.
Figure 5: Phylogenetic tree based on 16S rRNA sequence. Reference strain CP003069 was included

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Head-to-head comparison of these genotyping methods

Genotyping results were obtained successfully from one single round PCR with the optimised MISSR-PCR, ERIC-PCR and RAPD system, respectively. Though all the fingerprinting maps were clear and scorable, the MISSR-PCR system could generate higher polymorphism when compared to ERIC-PCR and RAPD system [28 bands vs 25 bands, [Figure 1], [Figure 3]a and [Figure 4]a. When these fingerprinting maps were used for genotyping and genetic tracing, the ERIC-PCR system could only differentiate toxigenic/nontoxigenic strains and local/foreign strains, while the O1 serogroup strains and O139 serogroup strains, as well as the O1 serogroup strains and non-O1/non-O139 serogroup strains, could not be separated. The RAPD system could overcome the weakness of ERIC-PCR system by clearly separating the O1 serogroup strains and O139 serogroup strains, though its ability to distinguish O1 serogroup strains from non-O1/non-O139 serogroup strains was still week. In addition, the RAPD system could generate a faint 320bp marker for toxigenic strains [Figure 4]a while no specific marker was generated from ERIC-PCR system. For the 16S rRNA direct sequencing, there was no genotyping ability at all as shown in the phylogenetic tree [Figure 5], which may be due to the high sequence similarity in the same species.

However, in huge contrast, the MISSR-PCR system showed significantly higher genotyping ability [Figure 2]a. First, the toxigenic and nontoxigenic strains could be separated clearly which was important for the quick response and control of the cholera epidemics. Second, the O1 serogroup strains and the O139 serogroup strains could also be separated in the dendrogram. Third, the local and foreign strains could be separated which would be useful in the epidemiology study. The forth advantage was that the nontoxigenic O1 serogroup strains could be separated from the nontoxigenic non-O1/non-O139 serogroup strains. Moreover, a specific 350 bp band was generated in the toxigenic strains [Figure 1], right panel], which was helpful for the quick identification of toxigenic strains. Therefore, the MISSR-PCR system was more powerful in Vibrio cholerae detection and genotyping when compared to the other methods.


 ~ Discussion Top


In the current study, the optimization and comparison of four genotyping methods (including MISSR-PCR, ERIC-PCR, RAPD, and 16S rRNA) for Vibrio cholerae was described. Results showed that the MISSR-PCR system could generate the highest polymorphic fingerprinting map and displayed the best discriminatory ability. As the cholera epidemic continues, the MISSR-PCR system would play an important role in the diagnosis, monitoring and control of Vibrio cholerae.

The MISSR-PCR fingerprinting method generated clear fingerprinting profiles with high polymorphism. Initially when the regular 22–24 mer primers were used for the MISSR-PCR, smear (for primer MISSR_GC6 especially) or limited bands (for other primers) were generated due to high GC content or high specificity of the primers (data not shown), which was not appropriate for the genotyping. Then the MISSR-PCR system was optimised by shortening the length of primers and adjusting the concentration of Mg 2+, dNTPs and primer. After several rounds of optimisation, the MISSR-PCR system was established with the optimised conditions using a 12mer primer MISSR_GA5 which could generate 28 bands varying from 150–3000 bp [Figure 1]. The fingerprinting map generated was clear and polymorphic, which was highly suitable for genotyping and genetic tracing.

A head-to-head comparison was conducted among these four genotyping methods, which showed distinct discriminatory ability in the cluster analysis. The dendrogram was generated by cluster analysis using the average linkage between groups' method. Though all of above methods could generate polymorphic fingerprinting maps, their discriminatory abilities were different. Unlike ERIC-PCR, RAPD or 16S rRNA which could not separate different serogroups strains as described above, MISSR-PCR could separate toxigenic/nontoxigenic strains, local/foreign strains, and O1/O139/non-O1/non-O139 serogroup strains with clear cluster profiles, indicating that the MISSR-PCR could serve as a powerful genotyping method. To the best of our knowledge, this is the first head-to-head comparison of these detecting and genotyping methods for Vibrio cholerae.

Moreover, a comparison between this MISSR-PCR system and the previously described ISSR-PCR method invented by G. Balakrish Nair and J. Nagaraju group in India [11] was also conducted. Till now, their method is the only ISSR-PCR-based assay developed for genotyping of Vibrio cholerae. In their study, they found that the ISSR-PCR-based phylogeny was consistent with the classification of Vibrio cholerae based on serological methods and toxigenic/non-toxigenic strains could be separated clearly. However, the fingerprinting profiles they generated were either less polymorphic [16 bands for primer (GA)8T] or in a narrower range [190–1300 bp for primer C (GA)7] compared to the MISSR-PCR system (28 bands varying from 150–3000 bp). In addition, the O1 serogroup strains and the O139 serogroup stains were in the same cluster and could not be separated on the dendrogram they generated, indicating their weak discriminative ability in Vibrio cholerae genotyping. In contrast, the MISSR-PCR system showed much better genotyping ability by separating Vibrio cholerae strains into clear sub-clusters as described above. Therefore, the MISSR-PCR is also superior to this traditional ISSR-PCR-based method.

One limitation of the current study is the quantity of the toxigenic strains included for analysis. Only 14 toxigenic strains were collected for genotyping, which are much less than non-toxigenic strains. Future study with more toxigenic strains is needed to further evaluate the MISSR-PCR system.

In conclusion, the novel MISSR-PCR system was optimized and established successfully. When compared to traditional ISSR-PCR, ERIC-PCR, RAPD and 16S rRNA evolutionary clock, the novel MISSR-PCR system generated the highest polymorphism and showed the clearest genotyping in one single PCR reaction. Thus, the MISSR-PCR could serve as a simple, quick, reliable and cost-effective tool in genotyping and epidemiological study of Vibrio cholerae and play a vital role in the diagnosis, monitoring and control of variable pathogens in food.


 ~ Acknowledgments Top


The authors thank Samantha Chen for proof-reading. This work was supported by grants from General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China (grant#2009IK206). The authors have declared that no conflict of interest exists.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04