TECHNICAL FIELD 
       [0001]    The present invention relates to the field of biotechnology, more specifically, to primers for identifying serotypes of  Shigella flexneri  and to multiplex amplifications using the same. 
       BACKGROUND OF THE INVENTION 
       [0002]      Shigella  species are the major pathogenic bacteria causing bacterial diarrhea in developing countries, which infect 164.7 million people and lead to 0.11 million deaths per year, most of which are children under 5 years old (Kotloff, K. L., J. P. Winickoff, B. Ivanoff, J. D. Clemens, D. L. Swerdlow, P. J. Sansonetti, G. K. Adak, and M. M. Levine. 1999. Global burden of  Shigella  infections: implications for vaccine development and implementation of control strategies. Bull World Health Organ 77:651-66). Among the four serogroups of  Shigella, Shigella flexneri  is the predominant serogroup that affects the low-income population. 
         [0003]      Shigella flexneri  is further classified into various serotypes according to the different 0-antigen structures. To date, at least 15 serotypes have been reported, i.e. 1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, X, Xv, Y, and F6 (Simmons, D. A., and E. Romanowska. 1987. Structure and biology of  Shigella flexneri  O antigens. J. Med. Microbiol. 23:289-302; Stagg, R. M., S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novel glucosyltransferase involved in O-antigen modification of  Shigella flexneri  serotype 1c. J. Bacteriol. 191:6612-7; Ye, C., R. Lan, S. Xia, J. Zhang, Q. Sun, S. Zhang, H. Jing, L. Wang, Z. Li, Z. Zhou, A. Zhao, Z. Cui, J. Cao, D. Jin, L. Huang, Y. Wang, X. Luo, X. Bai, P. Wang, Q. Xu, and J. Xu. 2010. Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of  Shigella flexneri  . J. Clin. Microbiol. 48:419-26). Serotyping has long been use to characterize isolates for epidemiological purposes 
         [0004]    The LPSs of all  Shigella flexneri  serotypes except F6 have the same polysaccharide backbone consisting of repeating tetrasaccharide units, and serotype Y has the basic tetrasaccharide backbone (Simmons, D. A., and E. Romanowska. 1987. Structure and biology of  Shigella flexneri  O antigens. J. Med. Microbiol. 23:289-302). Modification by glycosylation and/or acetylation of different sugars on the backbone gives rise to various type-specific antigenic determinants (e.g., I, II, III, IV, V, and VI), group-specific antigenic determinants (e.g., 3,4; 6; and 7,8), and antigenic determinant 1c (Stagg, R. M., S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novel glucosyltransferase involved in O-antigen modification of  Shigella flexneri  serotype 1c. J. Bacteriol. 191:6612-7). 
         [0005]    Three genes (gtrA, gtrB, and gtr [type] ) are responsible for glycosylation modifications on  Shigella flexneri . The first two genes are highly homologous and interchangeable, whereas the third gene gtr [type]  is unique, and encodes serotype-specific glycosyltransferases (Allison, G. E., and N. K. Verma. 2000. Serotype-converting bacteriophages and O-antigen modification in  Shigella flexneri . Trends. Microbiol. 8:17-23; Stagg, R. M., S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novel glucosyltransferase involved in O-antigen modification of  Shigella flexneri  serotype 1c. J. Bacteriol. 191:6612-7). Gtr genes specific to type-antigens I, II, IV and V, group antigen 7,8 and antigen 1c are gtrI, gtrII, gtrIV, gtrV, gtrX and gtrIC, respectively (Adams, M. M., G. E. Allison, and N. K. Verma. 2001. Type IV O antigen modification genes in the genome of  Shigella flexneri  NCTC 8296. Microbiology 147:851-60; Adhikari, P., G. Allison, B. Whittle, and N. K. Verma. 1999. Serotype 1a O-antigen modification: molecular characterization of the genes involved and their novel organization in the  Shigella flexneri  chromosome. J Bacteriol 181:4711-8; Guan, S., D. A. Bastin, and N. K. Verma. 1999. Functional analysis of the O antigen glucosylation gene cluster of  Shigella flexneri  bacteriophage SfX. Microbiology 145: 1263-73; Huan, P. T., D. A. Bastin, B. L. Whittle, A. A. Lindberg, and N. K. Verma. 1997. Molecular characterization of the genes involved in O-antigen modification, attachment, integration and excision in  Shigella flexneri  bacteriophage SfV. Gene 195:217-27; Mavris, M., P. A. Manning, and R. Morona. 1997. Mechanism of bacteriophage SfII-mediated serotype conversion in  Shigella flexneri . Mol Microbiol 26:939-50; Stagg, R. M., S. S. Tang, N. I. Carlin, K. A. Talukder, P. D. Cam, and N. K. Verma. 2009. A novel glucosyltransferase involved in O-antigen modification of  Shigella flexneri  serotype 1c. J Bacteriol 191:6612-7). The gtr genes are carried by prophages integrated in the genome of host bacteria. O-acetylation, which confers group-antigen 6 and/or type-antigen III on strains of serotypes 1b, 3a, 3b and 4b, is mediated by the oac gene carried in bacteriophage Sf6 (Clark, C. A., J. Beltrame, and P. A. Manning. 1991. The oac gene encoding a lipopolysaccharide O-antigen acetylase maps adjacent to the integrase-encoding gene on the genome of  Shigella flexneri  bacteriophage Sf6. Gene 107:43-52; Verma, N. K., J. M. Brandt, D. J. Verma, and A. A. Lindberg. 1991. Molecular characterization of the O-acetyl transferase gene of converting bacteriophage Sf6 that adds group antigen 6 to  Shigella flexneri . Mol. Microbiol. 5:71-5). Strains of different serotypes carry one or more serotype-specific prophages that encode different specific 0-antigen modifications genes (see  FIG. 1 ). 
         [0006]    Currently, the method for routinely identifying  Shigella flexneri  serotypes is the slide agglutination method using rabbit-originated antiserum raised against  Shigella flexneri -specific type- and group-factors. Commercially available diagnostic sera have been widely used in microbiology laboratories. However, such a method has some disadvantages. Firstly, to identify the serotype of one  Shigella  strain, the slide agglutination method requires as many as 10 reaction tests, using antibodies against type-antigens I, II, III, IV, V and VI, antibodies against group-antigens 3,4 and 7,8, and the monoclonal antibody against serotype 1c (MASF1c), respectively (Stagg, R. M., P. D. Cam, and N. K. Verma. 2008. Identification of newly recognized serotype 1c as the most prevalent  Shigella flexneri  serotype in northern rural Vietnam. Epidemiol Infect 136:1134-40; Talukder, K. A., Z. Islam, M. A. Islam, D. K. Dutta, A. Safa, M. Ansaruzzaman, A. S. Faruque, S. N. Shahed, G. B. Nair, and D. A. Sack. 2003. Phenotypic and genotypic characterization of provisional serotype  Shigella flexneri  1c and clonal relationships with 1a and 1b strains isolated in Bangladesh. J Clin Microbiol 41:110-7; Ye, C., R. Lan, S. Xia, J. Zhang, Q. Sun, S. Zhang, H. Jing, L. Wang, Z. Li, Z. Zhou, A. Zhao, Z. Cui, J. Cao, D. Jin, L. Huang, Y. Wang, X. Luo, X. Bai, P. Wang, Q. Xu, and J. Xu. 2010. Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of  Shigella flexneri . J Clin Microbiol 48:419-26). Secondly, visual assessment of the slide agglutination reactions may deliver incorrect readings. Thirdly, expensive antiserum kits limit the application of such a method in laboratories in developing countries. 
         [0007]    Therefore, development of a rapid, specific identification method based on biomolecular technologies such as PCR will be significant for timely and accurate identification of Shigella flexneri serotypes. 
       SUMMARY OF THE INVENTION 
       [0008]    One objective of the present invention is to provide a set of primers for identifying Shigella flexneri serotypes so as to identify  Shigella flexneri  serotypes by multiplex amplification, in acknowledgement of the O-antigen modification genes of  Shigella flexneri.    
         [0009]    Another objective of the present invention is to provide the use of the primers, in particular, for identification of  Shigella flexneri  serotypes and for preparation of agents for identifying  Shigella flexneri  serotypes. 
         [0010]    Yet another objective of the present invention is to provide a method for identifying  Shigella flexneri  serotypes by multiplex amplification using the primers. 
         [0011]    Still another objective of the present invention is to provide agents for identifying  Shigella flexneri  serotypes, such as a kit. 
         [0012]    In one aspect of the present&#39; invention, a set of primers for multiplex amplification used for identification of  Shigella flexneri  serotypes is designed based on genes gtrI, gtrII, oac, gtrIV, gtrV, gtrX, wzx 1-5  and gtrIC. The set of primers includes amplification primers for identifying target genes gtrI, gtrII, oac, gtrIV, gtrV, gtrX, wzx 1-5  and gtrIC. In a particular embodiment of the present invention, amplification primers having specificity and a common annealing temperature are designed based on known sequences of genes gtrI, gtrII, oac, gtrIV, gtrV, gtrX, wzx 1-5  and gtrIC, and more specifically, are the following amplification primers of the present invention: SEQ ID Nos. 1 and 2, for amplification of the gtrl gene fragment; SEQ ID Nos. 3 and 4, for amplification of the gtrII gene fragment; SEQ ID Nos. 5 and 6, for amplification of the oac gene fragment; SEQ ID Nos. 7 and 8, for amplification of the gtrIV gene fragment; SEQ ID Nos. 9 and 10, for amplification of the gtrV gene fragment; SEQ ID Nos. 11 and 12, for amplification of the gtrX gene fragment; SEQ ID Nos. 13 and 14, for amplification of the wzx 1-5  gene fragment; and SEQ ID Nos. 15 and 16, for amplification of the gtrIC gene fragment. 
         [0013]    The primers of the invention can be used for qualitative identification of  Shigella flexneri  serotypes. All currently known serotypes can be successfully identified, except for serotype Xv, which cannot be distinguished from serotype X. 
         [0014]    According to a preferred embodiment of the present invention, the set of primers for identifying  Shigella flexneri  serotypes provided by the present invention may further include a primer pair specific to F6. The average isolation rate of serotype F6 in Asia is 6%, ranging from 0% (China) to 15% (Pakistan) (von Seidlein, L., D. R. Kim, M. Ali, H. Lee, X. Wang, V. D. Thiem, G. Canh do, W. Chaicumpa, M. D. Agtini, A. Hossain, Z. A. Bhutta, C. Mason, 0. Sethabutr, K. Talukder, G. B. Nair, J. L. Deen, K. Kotloff, and J. Clemens. 2006. A multicentre study of  Shigella  diarrhoea in six Asian countries: disease burden, clinical manifestations, and microbiology. PLoS Med 3:e353). It is necessary to introduce an F6-specific singleplex PCR for countries where F6 is prevalent. However, since the multiplex amplification primers of the present invention already contain 8 pairs of primers, adding an additional pair of primers increases the difficulty in optimizing the multiplex PCR. Thus, one has to be cautious when interpreting the amplification-negative strains in the multiplex PCR as serotype F6. According to a preferred embodiment of the present invention, a singleplex PCR is employed to confirm F6, and the primer pair for identifying F6 is SEQ ID Nos. 17 and 18 for amplification of the wzx 6  gene fragment. Furthermore, as the strains have been verified with polyvalent antisera for  Shigella  and serogroup B prior to the multiplex PCR, the probability of false-positive F6 is low. 
         [0015]    In another aspect, the present invention further provides the use of the primers, particularly including the use of the primers for identification of  Shigella flexneri  serotypes and the use of the primers for preparation of agents for identifying  Shigella flexneri  serotypes. 
         [0016]    Based on the use of the primers for identifying  Shigella flexneri  serotypes provided by the present invention, the present invention also establishes a set of rapid and sensitive methods ready for high-throughput application, for identifying  Shigella flexneri  serotypes by amplification. In particular, the method is a multiplex amplification identification method, comprising carrying out identification by multiplex amplification using the primers of the present invention, wherein the amplification is preferably polymerase chain reaction (PCR). Since the multiplex amplification technique involves multiple pairs of primers and multiple pairs of templates as compared with conventional amplification techniques, it is influenced by more factors and thus has a higher chance to give mismatched amplification products. Therefore, the design of the primers is crucial. In the present invention, on the basis of the designed multiple pairs of primers, the reaction system and conditions, especially the annealing temperature, are preferably optimized. In PCR, the PCR system is composed of heat-resistant DNA polymerases, primers, deoxynucleotides, DNA templates to be amplified, and a buffer. The present invention provides a preferred PCR reaction which is performed under the following conditions: pre-denaturation at 95° C. for 15 min; 30 cycles of: denaturation at 94° C. for 30 s, annealing at 55° C. for 90 s, and extension at 72° C. for 60 s; and final extension at 72° C. for 10 min. 
         [0017]    The multiplex amplification-based identification method of the present invention preferably further includes performing a qualitative analysis after the amplification. The method for the qualitative analysis may be those known to a person skilled in the art, e.g., a method including visualization of amplification products using gel electrophoresis, in which case a person skilled in the art can determine the gel and gel concentrations to be used, according to the sizes of the amplification products. 
         [0018]    In order to validate the effectiveness of the method of the present invention, in particular embodiments of the present invention, the primers of the present invention were used to conduct amplification on strains representing 14 currently common  Shigella flexneri  serotypes by multiplex PCR, and 358  Shigella flexneri  strains of various serotypes were analyzed. The multiplex PCR results for the assayed strains have a concordance of 97.8% with those obtained by the slide agglutination method. In order to assess the specificity of the primers of the present invention, 50 non flexneri strains, including other  Shigella  serogroups and enteric pathogens, were also examined in particular embodiments of the present invention. These bacteria were all negative in the amplification, proving that the specificity of the method of the present invention is 100%. These results demonstrate that timely and accurate identification of  Shigella flexneri  serotypes can be achieved by using the primers and the amplification-based identification method of the present invention. 
         [0019]    According to the use of the primers in the preparation of agents for identifying  Shigella  serotypes as provided in the present invention, the identification agents of the present invention are preferably used to assay isolated  Shigella flexneri  strains. According to particular embodiments of the present invention, the identification agents may be a kit. The identification agents may be used to assay  Shigella flexneri  or assay  Shigella flexneri -containing samples used for identification and diagnosis. In the present invention, the identification agents are preferably used to assay excrement, intestinal effusions, and vomit. The multiplex amplification primers and the multiplex amplification method of the present invention are highly specific, can be used alone as a rapid identification method to assay enriched or unenrinched samples, such as ex vivo samples from patients (for example, excrement, effusions, vomit, etc.) and water, soil, food, and cosmetics that may carry  Shigella flexneri , and can achieve rapid diagnosis and identification of  Shigella flexneri  serotypes. 
         [0020]    The present invention further provides a kit for identifying  Shigella flexneri  serotypes, which contains the primers of the present invention. The kit may further comprise target-detecting probes for detecting target genes, and the probes are for example SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25 and SEQ ID No.26. The kit of the present invention can be used to qualitatively identify  Shigella flexneri  serotypes. 
         [0021]    Among  Shigella flexneri , serotypes X and Xv have the same amplification pattern (wzx 1-5  and gtrX). Thus, they are classified as the same serotype Xv or X by the multiplex PCR method. Serotype Xv has been one of the most prevalent serotypes in China since 2002 (Ye, C., R. Lan, S. Xia, J. Zhang, Q. Sun, S. Zhang, H. Jing, L. Wang, Z. Li, Z. Zhou, A. Zhao, Z. Cui, J. Cao, D. Jin, L. Huang, Y. Wang, X. Luo, X. Bai, P. Wang, Q. Xu, and J. Xu. 2010. Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of  Shigella flexneri . J Clin Microbiol 48:419-26), which was initially designated as 4c as it can react with both anti-type IV sera and anti-group 7,8 sera (Pryamukhina, N. S., and N. A. Khomenko. 1988. Suggestion to supplement  Shigella flexneri  classification scheme with the subserovar  Shigella flexneri  4c: phenotypic characteristics of strains. J Clin Microbiol 26:1147-9). However, since factors responsible for type IV antigenic determinant have not been clarified (Ye, C., R. Lan, S. Xia, J. Zhang, Q. Sun, S. Zhang, H. Jing, L. Wang, Z. Li, Z. Zhou, A. Zhao, Z. Cui, J. Cao, D. Jin, L. Huang, Y. Wang, X. Luo, X. Bai, P. Wang, Q. Xu, and J. Xu. 2010. Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of  Shigella flexneri . J Clin Microbiol 48:419-26), it is currently impossible to develop a PCR method to identify serotype Xv. In order to differentiate between serotypes Xv and X, an additional slide agglutination reaction using anti-type IV serum would be necessary. 
         [0022]    Above all, according to the present invention, specific primers with a common annealing temperature used for identifying  Shigella flexneri  serotypes have been designed, and a multiplex PCR identification method based on the genes of  Shigella flexneri  O antigen-modification enzymes has been established. The multiplex PCR method of the present invention may be employed for biomolecular identification of  Shigella flexneri  serotypes, and the majority of currently known serotypes (14 out of 15) can be easily and specifically identified in only one reaction. In comparison with the conventional slide agglutination method (requiring as many as 10 independent reactions), the multiplex PCR method of the present invention is time-saving, does not require expensive antisera, and is especially suitable for high-throughput identification. With this method, identification of 96 samples can be completed within 3.5 hours and costs only 25% of that of the slide method. The amplification primers and the multiplex amplification method of the present invention may be used to complement the serum agglutination method for identification of  Shigella flexneri  serotypes, and are practically significant and highly valuable for timely and accurate identification of serotypes of clinical isolates of pathogenic bacteria and for prevention and treatment of shigellosis. 
     
    
     
       DESCRIPTION OF THE DRAWINGS 
         [0023]      FIG. 1  shows the chemical composition and genes responsible for specific modifications of the O antigens of various  Shigella flexneri  serotypes. 
           [0024]      FIG. 2  shows the multiplex PCR amplification results of reference strains of 14 serotypes. The results were obtained by using the method of the present invention, wherein M1 and M2 are 150 by and 100 by DNA Ladder Markers (purchased from TaKaRa, Japan), respectively. 
           [0025]      FIG. 3  shows the specificity assay results of 50 non- Shigella flexneri  strains. In the results, M: marker; C: positive control; 1-2:  Shigella sonnei , phases I, II; 3-14:  Shigella dysenteriae  serotypes 1-12; 15-32:  Shigella boydii  serotypes 1-18; 33-34: Enteroaggregative  E. coli;  35-37: Enterohemorrhagic  E. coli  0157:H7; 38: Enteroinvasive  E. coli;  39: Enteropathogenic  E. coli;  40: Enterotoxigenic  E. coli;  41: Uropathogenic  E. coli;  42-43:  E. coli  K12; 44: L monocytogenes; 45:  V. cholera;  46:  Salmonella paratyphi  A; 47-48:  Salmonella paratyphi  B; 49:  Yersinia enterocolitica;  50:  Salmonella choleraesuis.    
       
    
    
     DETAILED DESCRIPTION 
       [0026]    The present invention will be further described with reference to the following examples and the figures, for better understanding of the invention. The examples are only illustrative and are not in any way limiting the scope of the present invention. In the examples, any experimental methods without detailed conditions specified are conventional methods with conventional conditions well known in the art or conditions recommended by the manufacturer. 
       EXAMPLES 
     Materials and Methods 
       [0027]    Strains: 14 known  Shigella flexneri  serotypes (except serotype 5b, see Table 1 for details) were used in the examples to establish the conditions for performing the multiplex PCR method. 358  Shigella flexneri  strains in total (Table 2) were used to evaluate the effectiveness of the multiplex PCR method of the present invention. In order to examine the cross-reactions of the primers in this study, 50 strains from different genera were examined ( S. Sonnei  (n=2),  S. dysenteriae  (n=12, including all 12 serotypes),  S. boydii  (n=18, including all 18 serotypes), Enteroaggregative  E. coli  (n=2), Enterohemorrhagic  E. coli  O 157:H7 (n=3), Enteroinvasive  E. coli  (n=1), Enteropathogenic  E. coli  (n=1), Enterotoxigenic  E. coli  (n=1), Uropathogenic  E. coli  (n=1),  E. coli  K12 (n=2),  L. monocytogenes  (n=1),  V cholera  (n=1),  Salmonella paratyphi  A (n=1),  Salmonella paratyphi  B (n=2),  Yersinia enterocolitica  (n=1), and  Salmonella choleraesuis  (n=1)). The serotypes of all the  Shigella flexneri  strains were confirmed with polyvalent antisera (purchased from Denka Seiken, Japan) and monoclonal antibodies (purchased from Reagensia AB, Sweden). All Chinese strains used in the examples were isolated from diarrhea patients in Chinese and were preserved in the Microbiology Laboratory, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC). Other strains were purchased from National Collection of Type Cultures (NCTC), UK. 
       Preparation of DNA Templates 
       [0028]    DNA templates were directly obtained from bacteria clones by the boiling method. A single colony from an overnight culture on an LB plate was firstly put into 30 μl distilled water, boiled at 100° C. for 10 min, placed into an ice bath for 5 min, and centrifuged at 13,000 ×g, 4° C. for 10 min. The supernatant was used as the templates for PCR amplification. 
       PCR Primers 
       [0029]    The PCR primers used in the examples are listed in Table 3, the primers for amplification of the wzx gene were designed with reference to Yayue Li et al. (Li, Y., B. Cao, B. Liu, D. Liu, Q. Gao, X. Peng, J. Wu, D. A. Bastin, L. Feng, and L. Wang. 2009. Molecular detection of all 34 distinct O-antigen forms of Shigella. J Med Microbiol 58:69-81), and other primers were designed according to the sequences of  Shigella flexneri  serotype-specific genes gtrI, gtrIC, gtrII, oac, gtrIV, gtrV, and gtrX. 
         [0030]    All primers were synthesized by Sangon Biotech (Shanghai) as commissioned, and dissolved in TL buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) to have a final concentration of 50 μM. 
       PCR Amplification and Identification 
       [0031]    Multiplex PCRs were carried out using a QIAGEN Multiplex PCR Kit (QIAGEN). Each PCR reaction mixture comprises 1×PCR Master Mix (containing HotStarTaq DNA polymerase, Multiplex PCR buffer, and dNTP Mix), primers (0.2 μM each) and 3 μl DNA templates, and is 50 μl in total. In the examples, PCR amplification was performed based on the PCR conditions provided by the kit instructions in which some multiplex PCR cycling parameters were further optimized, Le, pre-denaturation at 95° C. for 15 min; totally 30 cycles of denaturation at 94° C. for 30s, annealing at 55° C. for 90 s, extension at 72° C. for 60 s; and final extension at 72° C. for 10 min. The amplification was performed in a thermocycler from SENSO (Germany). 5 microliter of amplification products was mixed with loading buffer, electrophoresed on a 1.5% agarose gel, and visualized using EB staining for results. Where necessary, the PCR products were directly sequenced or cloned into a pMD20-T TA cloning vector (TaKaRa, Japan) for cloning and sequencing. 
       Experimental Results 
       [0032]    In the examples, reference strains were firstly subjected to singleplex PCR with. the primers, and as a results, every pair of primers can give the expected fragments after amplification, namely 783 by (wzx 1-5 ), 1122 by (gtrI), 518 by (gtrIC), 1268 by (gtrII), 604 by (oac), 378 bp (gtrIV), 905 by (gtrV) and 425 by (gtrX), respectively. Sequencing of the PCR amplification products proved that the amplified fragments were correct. Afterwards, multiplex PCR was carried out according to the protocols described above in Materials and Methods, in which we tried different annealing temperatures from 54° C. to 63° C. and found that the highest yield was obtained at 55° C. The results showed that different serotypes have different amplification patterns (see  FIG. 2  and Table 1). The expected specific PCR products were obtained from the amplification of each serotype. The amplified fragments of different sizes can be well resolved on a 1.5% gel, and with two different DNA markers, each PCR product can be correctly identified by size. The amplification patterns of the reference strains are as follows: 1a (wzx 1-5 , gtrI), 1b (wzx 1-5 , gtrI, oac), 1c (wzx 1-5 , gtrI, gtrIC), 2a (wzx 1-5 , gtrII), 2b (wzx 1-5 , gtrII, gtrX), 3a (wzx 1-5 , oac, gtrX), 3b (wzx 1-5 , oac), 4a (wzx 1-5 , gtrIV), 4b (wzx 1-5 , gtrIV, oac), 5a (wzx 1-5 , gtrV), X/Xv (wzx 1-5 , gtrX) and Y (wzx 1-5 ). wzx 1-5  may also be used as a reference gene for PCR reactions since it is present in all  Shigella flexneri  serotypes except serotype F6, F6 is negative in the amplification as its O-antigen synthesizing genes are completely different from those of other serotypes, and does not have any genes for modifications (Cheah, K. C., D. W. Beger, and P. A. Manning. 1991. Molecular cloning and genetic analysis of the rfb region from  Shigella flexneri  type 6 in  Escherichia coli  K-12. FEMS Microbiol Lett 67:213-8; Simmons, D. A., and E. Romanowska., 1987. Structure and biology of  Shigella flexneri  O antigens. J Med Microbiol 23:289-302). 
         [0033]    In the examples, we attempted to incorporate a pair of specific primers for F6 in the reaction. However, the additional pair of primers increases the difficulty in optimization of the multiplex PCR since the reaction already contains 8 pairs of primers. Hence, no good results were obtained after experimentation with 3 pairs of such primers (designed according to the sequence of F6-specific O-antigen gene wzx 6 ), and one should be cautious when interpreting a strain negative in the amplification as serotype F6. According to preferred embodiments of the present invention, a singleplex PCR was used to confirm F6, the primers for which can be seen in Table 3, and the length of the amplified product is 739 bp. Furthermore, since all strains have been verified with polyvalent antisera for  Shigella  and serogroup B, possibility of a false-positive F6 is very low. 
         [0034]    It is noteworthy that the primers for amplification of the oac gene were designed based on the conserved regions in oac and oac 1b  (which share 88% identity) to guarantee both can be amplified. Although a serotype 5b strain was not available, it can be predicted that the amplification pattern of serotype 5b is 905 by (gtrV) and 425 by (gtrX) according to the 0 antigen-modification characteristics of this serotype. As there are not any typical strains of serotype 1 c, a serotype F 1 strain (untypable), 06HN081 (this strain can react with the 1c-specific monoclonal antibody MASF 1c), was used in the present invention to detect the serotype 1c-specific gene gtrIC. As compared to typical 1c strains (which react exclusively with MASF1c), this F1 strain (untypable) can react with both MASF1c and group 6 antisera. Accordingly, the genes gtrIC and oac that encode the antigen of MASF1c and the group 6 antigen were both identified as positive. These results indicate that this F1 (untypable) strain is converted from serotype 1b having its O-antigen modified by GtrIC, and is therefore a new serotype (see Table 1). This new serotype has the same serological reaction pattern as the strain of serotype 7b reported in Forster et al., and thus the serotype of 06HN081 should be 7b. It is to be noted that 1c is designated in Forster et al. as 7a (Foster, R. A., N. I. Carlin, M. Majcher, H. Tabor, L. K. Ng, and G. Widmalm. Structural elucidation of the O-antigen of the  Shigella flexneri  provisional serotype 88-893: structural and serological similarities with  S. flexneri  provisional serotype Y394 (1c). Carbohydr Res 346:872-6). Hence, these two new serotypes can be identified by the multiplex PCR of the present invention. The present invention further provides the use of the primers, the multiplex PCR method, and the kit for identifying these two new serotypes. 
         [0035]    Serotypes X and Xv have the same amplification pattern (wzx 1-5  and gtrX). Thus, they are classified as the same serotype Xv/X by the multiplex PCR method. 
         [0036]    In order to evaluate the specificity of the primers, 50 non- Shigella flexneri  strains were tested in the examples, including other  Shigella  serogroups and enteric pathogens. These bacteria all showed negative after the amplification (see  FIG. 3 ), indicating the specificity of the method of the present invention is 100%. 
         [0037]    To determine whether the method of the present invention is applicable to all  Shigella flexneri  strains, and to assess the effectiveness of the method of the present invention, 358  Shigella flexneri  strains of various serotypes were analyzed in the examples (see Table 2). The multiplex PCR results of nearly all tested strains (except 8 strains) are consistent with the slide agglutination results, with a concordance rate of 97.8%. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Serotype characteristics of  S. flexneri  reference strains by  
               
               
                 agglutination and multiplex PCR 
               
             
          
           
               
                   
                   
                 Serum Agglutination Reaction 
                   
               
             
          
           
               
                   
                   
                 Type Antigen 
                 Group Antigen 
                 MASF 
                 Multiplex PCR 
               
             
          
           
               
                 Strain No. 
                 Serotype 
                 □ 
                 □ 
                 □ 
                 IV 
                 V 
                 □ 
                 3,4 
                 6 
                 7,8 
                 1c 
                 wzx 
                 gtrI 
                 gtrIC 
                 gtrII 
                 oac 
                 gtrIV 
                 gtrV 
                 gtrX 
               
               
                   
               
               
                 2000019 
                 1a 
                 + 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 + 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                 1997020 
                 1b 
                 + 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 + 
                 − 
                 − 
                 + 
                 + 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
               
               
                 06HN081 #   
                 — 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 + 
                 + 
                 + 
                 + 
                 − 
                 + 
                 − 
                 − 
                 − 
               
               
                 301 
                 2a 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
               
               
                 NCTC4 
                 2b 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 + 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
               
               
                 03HL12 
                 3a 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 + 
                 + 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 + 
               
               
                 2002110 
                 3b 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
               
               
                 NCTC9725 
                 4a 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
               
               
                 NCTC9726 
                 4b 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 + 
                 − 
                 − 
               
               
                 51247 
                 5a 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
               
               
                 2003036 
                 Y 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                 2001014 
                 X 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
               
               
                 2002017 
                 Xv 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
               
               
                 2000007 
                 F6 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
               
               
                   # Atypical F1 type, positive for both MASF1c and group 6 antiserum 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Correlation between the test results obtained from the multiplex PCR method and the 
               
               
                 slide agglutination method for 358  Shigella flexneri  strains of various serotypes. 
               
             
          
           
               
                   
                   
                   
                 Multiplex PCR 
               
               
                   
                 No. of 
                 Target Gene 
                 serotype 
               
             
          
           
               
                 Serotype 
                 Strains 
                 wzx 1-5   
                 gtrI 
                 gtrIC 
                 gtrII 
                 oac 
                 gtrIV 
                 gtrV 
                 gtrX 
                 classification 
               
               
                   
               
             
          
           
               
                 F1a 
                 25 
                 25 
                 25 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1a (25) 
               
               
                 F1b 
                 14 
                 14 
                 14 
                 0 
                 0 
                 14 
                 0 
                 0 
                 0 
                 1b (14) 
               
               
                 F2a 
                 55 
                 55 
                 0 
                 0 
                 55 
                 0 
                 0 
                 0 
                 0 
                 2a (55) 
               
               
                 F2b 
                 50 
                 50 
                 0 
                 0 
                 50 
                 0 
                 0 
                 0 
                 50 
                 2b (50) 
               
               
                 F3a 
                 10 
                 10 
                 0 
                 0 
                 0 
                 10 
                 0 
                 0 
                 10 
                 3a (10) 
               
               
                 F3b 
                 2 
                 2 
                 0 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 3b (2)  
               
               
                 F4a 
                 5 
                 5 
                 0 
                 0 
                 0 
                 0 
                 5 
                 0 
                 0 
                 4a (5)  
               
               
                 F4b 
                 5 
                 5 
                 0 
                 0 
                 0 
                 5 
                 5 
                 0 
                 0 
                 4b (5)  
               
               
                 F5a 
                 4 
                 4 
                 0 
                 0 
                 0 
                 1 
                 0 
                 4 
                 0 
                 5a (3), untypable 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 (1) 
               
               
                 Y 
                 36 
                 36 
                 0 
                 0 
                 5 
                 0 
                 0 
                 0 
                 0 
                 Y (31), 2a (5) 
               
               
                 Xv 
                 78 
                 78 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 78 
                 X or Xv (78) 
               
               
                 X 
                 69 
                 69 
                 0 
                 0 
                 2 
                 0 
                 0 
                 0 
                 69 
                 X or Xv (67), 2b 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 (2) 
               
               
                 F6 
                 5 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 F6 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 3  
               
             
             
               
                   
               
               
                 Primers Used in the Examples 
               
             
          
           
               
                   
                   
                 Length of 
                   
                   
               
               
                 Target 
                   
                 Amplified 
                 Serotype 
                 Accession 
               
               
                 Gene 
                 Primer sequence (5′→3′) 
                 Fragments (bp) 
                 Specificity 
                 no. 
               
               
                   
               
               
                 gtrI 
                 (F)CTGTTAGGTGATGATGGCTTAG 
                 1122 (SEQ ID 
                 1a, 1b, 1c 
                 AF139596 
               
               
                   
                 (SEQ ID No. 1) 
                 No. 19) 
                   
                   
               
               
                   
                 (R)ATTGAACGCCTCCTTGCTATGC 
                   
                   
                   
               
               
                   
                 (SEQ ID No. 2) 
                   
                   
                   
               
               
                   
               
               
                 gtrII 
                 (F)ATTTATTGTTATTGGGGGTGGTTG 
                 1268 (SEQ ID 
                 2a, 2b 
                 AF021347 
               
               
                   
                 (SEQ ID No. 3) 
                 No. 20) 
                   
                   
               
               
                   
                 (R)ATTTGTTCTTTATTTGCTGGTT 
                   
                   
                   
               
               
                   
                 (SEQ ID No. 4) 
                   
                   
                   
               
               
                   
               
               
                 oac 
                 (F)CTGTTCGGCTTTGAAAGTGCTG 
                 604 (SEQ ID 
                 1b, 3a, 3b, 
                 AF547987 
               
               
                   
                 (SEQ ID No. 5) 
                 No. 21) 
                 4b 
                   
               
               
                   
                 (R)CGTAGGCGTACATAGCAAGCAAAGA 
                   
                   
                   
               
               
                   
                 (SEQ ID No. 6) 
                   
                   
                   
               
               
                   
               
               
                 gtrIV 
                 (F)ATGTTCCTCCTTCTTCCTTT 
                 378 (SEQ ID 
                 4a, 4b 
                 AF288197 
               
               
                   
                 (SEQ ID No. 7) 
                 No. 22) 
                   
                   
               
               
                   
                 (R)TCCTGATGCTACCTTATCCA 
                   
                   
                   
               
               
                   
                 (SEQ ID No. 8) 
                   
                   
                   
               
               
                   
               
               
                 gtrV 
                 (F)AATACGATTCTCCTGGTGCTAAAC 
                 905 (SEQ ID 
                 5a, 5b 
                 U82619 
               
               
                   
                 (SEQ ID No. 9) 
                 No. 23) 
                   
                   
               
               
                   
                 (R)TAGGGCATTGCTTGTATCTTTCAT 
                   
                   
                   
               
               
                   
                 (SEQ ID No. 10) 
                   
                   
                   
               
               
                   
               
               
                 gtrX 
                 (F)AATGCTGGATGGGATAATCACCTT 
                 425 (SEQ ID 
                 2b, 3a, 5b, 
                 L05001 
               
               
                   
                 (SEQ ID No. 11) 
                 No. 24) 
                 X, Xv 
                   
               
               
                   
                 (R)GAGACGGCTTCTCCATGTTTTGCT 
                   
                   
                   
               
               
                   
                 (SEQ ID No. 12) 
                   
                   
                   
               
               
                   
               
               
                 wzx 1-5   
                 (F) CACTTGTTGGGTATGCTGG 
                 783 (SEQ ID 
                 1-5, X, Xv, 
                 AE005674 
               
               
                   
                 (SEQ ID No. 13) 
                 No. 25) 
                 Y 
                   
               
               
                   
                 (R) CCGGCAAACAGATTAGAAA 
                   
                   
                   
               
               
                   
                 (SEQ ID No. 14) 
                   
                   
                   
               
               
                   
               
               
                 gtrIC 
                 (F) AGGGAATGGCATTAGGGATCGG 
                 518 (SEQ ID 
                 1c 
                 FJ905303 
               
               
                   
                 (SEQ ID No. 15) 
                 No. 26) 
                   
                   
               
               
                   
                 (R) GCTGCAAGTGGTTTTTGTTGGA 
                   
                   
                   
               
               
                   
                 (SEQ ID No. 16) 
                   
                   
                   
               
               
                   
               
               
                 wzx 6   S   
                 (F) TTAAGAGCGATCATTTC 
                 739 (SEQ ID 
                 F6 
                 EU294165 
               
               
                   
                 (SEQ ID No. 17) 
                 No. 27) 
                   
                   
               
               
                   
                 (R) CCATCCAAGCGGACATT 
                   
                   
                   
               
               
                   
                 (SEQ ID No. 18) 
               
               
                   
               
               
                   S The primer pair for wzx 6  is used to conform serotype F6. 
               
             
          
         
       
     
         [0038]    The multiplex PCR method of the present invention may be employed to conduct biomolecular identification of  Shigella flexneri  serotypes in only one reaction, and can easily and specifically identify the majority of currently known serotypes (14 out of 15). in comparison with the conventional slide agglutination method (which requires as many as 10 independent reactions), the multiplex PCR method of the present invention is time-saving, does not require expensive antisera, and is especially suitable for high-throughput identification. Using this method, identification of 96 samples can he completed within 3.5 hours and costs only 25% of that of the slide agglutination method.
The present inventions relates to primers for identifying Shigella flexneri serotypes comprising the sequences of SEQ ID Nos. 1 and 2, SEQ ID Nos. 3 and 4, SEQ ID Nos. 5 and 6, SEQ ID Nos. 7 and 8, SEQ ID Nos. 9 and 10, SEQ ID Nos. 11 and 12, SEQ ID Nos. 13 and 14, SEQ ID Nos. 15 and 16. These primers are specific and have a common annealing temperature. The present invention further relates to a multiplex amplification-based identification method using the primers. The present invention further relates to the use of the primers for identifying Shigella flexneri serotypes for the preparation of identification agents. The present invention further relates to a kit for identifying Shigella flexneri comprising the above primers.