Patent Publication Number: US-2022220535-A1

Title: Oligonucleotides for detecting lactobacillus and method for detecting lactobacillus by using same

Description:
BACKGROUND 
     1. Technical Field 
     This disclosure relates to nucleic acids, probes, kits and methods for detection of organisms, including a strain of  Lactobacillus  sp. 
     2. Description of Related Art 
     Lactic acid bacteria (hereinafter, referred to as LAB) are bacteria that are capable of converting carbohydrate substrates into organic acids, mostly lactic acid, and producing a wide range of metabolites. LAB has been found to possess many health beneficial effects when administered to animals. For example, LAB is shown effective in helping with gut health, because of their capability in regulating host&#39;s gut microbiota, and found effective in preventing or treating abdominal pain, diarrhea, constipation, and bloating. 
     LAB is also shown to modulate host&#39;s mental and physical responses for psychological stress. Stress is one of the factors inducing mood disorders and neurochemical changes in both human and animals, and can lead to stress-induced disorders including anxiety, depression and irritable bowel syndrome. Therefore, stress-induced disorders can be treated by LAB as a supplement, in addition to the conventional psychiatric medicines, as LAB has been shown to have an influence on host gut-brain axis (GBA). For example,  Lactobacillus rhamnosusis  shown to alter functions of the central nerve system in healthy mice through vagus nerve, and the stressed rats supplemented with the probiotic strain  Bifidobacterium infantis  have reversed the behavioral deficits in forced swimming test (FST) and restored basal noradrenaline (NA) level in brain stem. 
     However, LAB constitutes a heterogeneous group and is found in diverse nutrient-rich habitats associated with plant and animal&#39;s matter, as well as in respiratory, gastrointestinal, and genital tracts of humans. Within the Firmicutes phylum, LAB members belong to the order  Lactobacillales  and comprise the following genera:  Aerococcus, Alloiococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus,  and  Weissella.  Among these,  Lactobacillus  is the largest genus of the LAB group, with over 100 species in total. For an instance, from the spontaneously fermented mustard products named “fu-tsai” and “suan-tsai,” which are traditionally prepared by the Hakka tribe of Taiwan, 500 LAB isolates were analyzed and identified as 119 representative strains belonging to 5 genera and 18 species, including  Enterococcus  (1 species),  Lactobacillus  (11 species),  Leuconostoc  (3 species),  Pediococcus  (1 species), and  Weissella  (2 species). 
     Furthermore, the probiotic properties of LAB are strain-specific, as these bacteria adapt to different environments and evolve accordingly. Therefore, it is necessary to be able to identify a specific strain of LAB from others, especially when the LAB strain has been discovered to possess certain health beneficial properties. For example, a LAB strain PS128 has been found to decrease serum corticosterone and alleviate a stress-induced disorder or a psychiatric disorder in a subject as demonstrated in EP Patent No. 2937424B1. The emotional calming effect of PS128 is found to extend to other non-human animals such as dogs (WO 2020/156418 A 1 ). The LAB strain PS128 also claims to treat functional gastrointestinal disorder such as constipation and functional dyspepsia (US10188684B2). The LAB strain PS128 is also shown to prevent or treat a movement disorder such as tic disorders and basal ganglia disorders by modulating dopamine neurotransmission in basal ganglia (WO 2018/014225 A1). Also, the LAB strain PS128 is shown to enhance physical strength and performance in a subject by increasing endurance and reducing muscle fatigue or muscle damage (WO 2020/156417 A1). 
     Hence, a method to specifically identify and discriminate a LAB strain from others is in need. 
     SUMMARY 
     The present disclosure provides a method for detecting a  Lactobacillus  species,  Lactobacillus plantarum  subsp.  plantarum  PS128 (hereinafter referred to as PS128). PS128 is deposited under DSMZ Accession No. DSM 28632. The method involves amplifying a nucleic acid sequence of PS128. The method further involves detecting the amplification product. The present disclosure also encompasses nucleic acids that can be used as primers to amplify a genomic nucleic acid sequence of PS128. In an embodiment, the nucleic acids used as primers hybridize to specific sequences of PS128 under nonarbitrary hybridization conditions. In an embodiment, the nucleic acid sequence of PS128 amplified by the primers is unique to the PS128 strain. The primers can be chemically synthesized oligonucleotides having the sequences 5′-TGTTGGGATGTTCTCTGCCT-3′ (SEQ ID NO: 1) or 5′-ACATTTACTGCGTTCTGTGC-3′ (SEQ ID NO: 2), or the corresponding complementary sequences. For example, the nucleic acid used as a primer has a sequence that contains at least 12, 13, 14, 15, 16, 17, 18 or 19 consecutive nucleotides, or the full length, of the above-identified sequences, as long as they can specifically hybridize to the nucleic acid sequence of PS128 and lead to an amplification product. In an embodiment, the amplification product amplified by the primers of the above-identified sequences is around 900 base pairs (bp), e.g., 890 bp, 895 bp, 900 bp, 905 bp, 910 bp, 915 bp, 920 bp, 925 bp and 930 bp. In another embodiment, the amplification product is 915 base pairs. 
     In an embodiment, the primers (or probes) can be provided in a detection kit. The kit may also include positive and negative controls for the above species. The positive control can be any sample that contains a target DNA to be amplified, including the bacteria themselves, at an amount over the detection limit. The negative control is a sample that does not contain the target DNA to be amplified. 
     In one embodiment, the amplification step of the method of the present disclosure is accomplished by polymerase chain reaction (PCR). In some embodiments, the amplification reaction may be selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), T7 polymerase mediated amplification, T3 polymerase mediated amplification, and SP6 polymerase mediated amplification. 
     In one embodiment, the amplification product is detected by gel electrophoresis, mass spectroscopy, or a single stranded DNA detection technique such as fluorescence resonance energy transfer (FRET). 
     In one aspect, provided are reaction mixtures. In some embodiments, the reaction mixtures comprise: (i) a sample comprising a nucleic acid template; and (ii) one or more oligonucleotide pairs configured to detect the presence or absence of a unique genomic sequence of PS128. In some embodiments, the oligonucleotide pairs detect the unique genomic sequence region of PS128. In some embodiments, the one or more oligonucleotide pairs comprise a forward primer having the sequence of SEQ ID NO: 1, or the complementary sequence of SEQ ID NO: 1, and a reverse primer of SEQ ID NO: 2, or the complementary sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid template comprises genomic DNA. In some embodiments, the reaction mixture further comprises a polymerase and dNTPs. 
     In another aspect, provided are kits. In some embodiments, the kits comprise an oligonucleotide pair that specifically identify the presence or absence of a unique genomic sequence of PS128. In some embodiments, the one or more oligonucleotide pairs comprise a forward primer having the sequence of SEQ ID NO: 1, or the complementary sequence of SEQ ID NO: 1, and a reverse primer of SEQ ID NO: 2, or the complementary sequence of SEQ ID NO: 2. 
     Other advantages and features of the present disclosure will become apparent from the following detailed descriptions of this disclosure. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a photograph of electrophoresis gel showing the amplification result of PCR using PS128 as the sample with a forward primer having the sequence of SEQ ID NO: 1 and a reverse primer having the sequence of SEQ ID NO: 2 (lane 2). Lane 1 shows the 100 bp ladders for estimating the size of PCR products. Lane 3 is a negative control using  L. plantarum  ATCC 14917 T  as the sample. 
         FIG. 2  is a photograph of electrophoresis gel showing the amplification result of PCR using PS128 (lane 2),  L. plantarum  299v (lane 3),  L. plantarum  LP-115 (lane 4),  L. plantarum  14D (lane 5),  L. plantarum  LP LDL  (lane 6),  L. plantarum  TWK-10 (lane 7),  L. plantarum  GL208 (lane 8), and  L. plantarum ATCC  14917 T  (lane 9) as the samples, respectively, with a forward primer having the sequence of SEQ ID NO: 1 and a reverse primer having the sequence of SEQ ID NO: 2. Lanes 1 and 10 show the 100 bp ladders for estimating the size of PCR products. 
         FIG. 3  is a photograph of electrophoresis gel showing the amplification result of PCR using PS128 (lane 2),  L. plantarum  299v (lane 3),  L. plantarum  LP-115 (lane 4),  L. plantarum  14D (lane 5),  L. plantarum  LP LDL  (lane 6),  L. plantarum  TWK-10 (lane 7), L. plantarum GL208 (lane 8) and L. plantarum ATCC 14917T (lane 9) as the samples, respectively, and a negative control using  Lactobacillus rhamnosus  GG (lane 10) as the sample with a forward primer having the sequence of SEQ ID NO: 3 and a reverse primer having the sequence of SEQ ID NO: 4. Lanes 1 and 11 show the 100 bp ladders for estimating the size of PCR products. The lanes 2 to 9 all show bands at 248 bp. 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer&#39;s specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (e.g., Green and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel, ed., Current Protocols in Molecular Biology, 1990-2017, John Wiley Interscience), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry and organic synthesis described below are those well-known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses. 
     The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). 
     Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term “nucleic acid” is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. 
     The phrase “nonarbitrary hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Nonarbitrary conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, the nonarbitrary conditions may be stringent conditions selected to be about 5 to 10° C. lower than the thermal melting point for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times of background, optionally 10 times of background hybridization. Exemplary conditions for stringent hybridization can be as following: 50% formamide, 5×saline-sodium citrate (SSC), and 1% sodium dodecyl sulfate (SDS), incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. 
     Nucleic acids that do not hybridize to each other under stringent conditions may still be substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. 
     A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. 
     The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under the given hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA). 
     The amplification of nucleic acids can be effected by the PCR method developed by Seiki et al. (Science, 230, 1350 (1985)). This method comprises preparing two oligonucleotides, one recognizing and hybridizing with the plus strand and the other recognizing and hybridizing with the minus strand at both ends of a specific nucleic sequence region to be detected, causing them to function as primers for the template-dependent nucleotide polymerization reaction against the sample nucleic acid in the single stranded form as a result of heat denaturation, separating the resulting double-stranded nucleic acid into single strands, and again allowing the same reaction to proceed. By repeating this serial procedure, the number of copies of the region between the two primers is increased so that said region can be detected. 
     The following specific examples are used for illustrating the present disclosure. A person skilled in the art can easily conceive the other advantages and effects of the present disclosure. The present disclosure can also be implemented by different cases enacted or application, and the details of the instructions can also be based on different perspectives and applications in various modifications and changes that do not depart from the spirit of the disclosure. 
     Many examples have been used to illustrate the present disclosure. The examples below should not be taken as a limit to the scope of this disclosure. 
     EXAMPLES 
     EXAMPLE 1. Identification of  Lactobacillus plantarum  subsp.  plantarum  PS128 by polymerase chain reaction (PCR) 
     PCR carried out with specific primers designed to amplify the unique genomic sequence region of PS128 was conducted to distinguish the bacteria with high sequence similarity. 
     The PCR of PS128 was carried out using 20 ng of DNA extracted from PS128, with reagents listed in Table 1 under the condition indicated in Table 2. DNAs extracted from this strain were used as templates. The obtained amplification products were electrophoresed, and the result was shown in  FIG. 1 , wherein the primers represented by SEQ ID NO: 1 and SEQ ID NO: 2 below were used. 
     
       
         
           
               
               
            
               
                   
                 Forward primer: 
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 5′-TGTTGGGATGTTCTCTGCCT-3′ 
               
               
                   
                   
               
               
                   
                 Reverse primer: 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 5′-ACATTTACTGCGTTCTGTGC-3′ 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Composition of the PCR reaction solution 
               
               
                 (25 μL per PCR tube) 
               
            
           
           
               
               
               
            
               
                   
                 PCR reagents 
                 Volume 
               
               
                   
                   
               
               
                   
                 Template DNA 
                  2.0 μL 
               
               
                   
                 (10 ng/μL) 
                   
               
               
                   
                 10X Ex Taq Buffer *   
                  2.5 μL 
               
               
                   
                 (Mg 2+  plus) 
                   
               
               
                   
                 dNTP (2.5 mM) 
                  2.0 μL 
               
               
                   
                 Forward primer (10 μM) 
                  2.0 μL 
               
               
                   
                 Reverse primer (10 μM) 
                  2.0 μL 
               
               
                   
                 TaKaRa Ex Taq *  (5 U/μL) 
                  0.2 μL 
               
               
                   
                 ddH 2 O 
                 14.3 μL 
               
               
                   
                   
               
               
                   
                   * Takara Bio U.S.A., Inc. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 PCR Conditions 
               
            
           
           
               
               
               
               
            
               
                   
                 Temperature 
                 Time  
                 Cycle 
               
               
                   
                   
               
               
                   
                 95° C. 
                  5 min 
                   
               
               
                   
                 95° C. 
                 30 sec 
                 30 cycles 
               
               
                   
                 59° C. 
                 30 sec 
                   
               
               
                   
                 72° C. 
                 60 sec 
                   
               
               
                   
                 72° C. 
                 10 min 
               
               
                   
                   
               
            
           
         
       
     
     As shown in  FIG. 1 , lane 1 represents DNA ladder (100-3000bp); lane 2 represents the amplification product with a size of around 900 bp by using PS128 DNA as the DNA template; and lane 3 represents the PCR result of  Lactobacillus plantarum  ATCC 14917 T , which is used as the negative control. 
     EXAMPLE 2. Specificity of the PCR method for detection of PS128 
     To test the specificity of the PCR method of the present disclosure, primers having sequences of SEQ. ID NOs: 1 and 2 were used to amplify other strains of  Lactobacillus plantarum.    
     A total of 7 different strains of  Lactobacillus plantarum  were obtained, as shown in Table 3 below. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Other tested  Lactobacillus plantarum  strains 
               
            
           
           
               
               
               
            
               
                   
                 
                   Lactobacillus  
                 
                   
               
               
                   
                   plantarum  strains 
                 Source 
               
               
                   
                   
               
               
                   
                 299v 
                 Probi AB, Sweden 
               
               
                   
                 LP-115 
                 Dupont, USA 
               
               
                   
                 14D 
                 Centro Sperimentale 
               
               
                   
                   
                 del Latte, Italy 
               
               
                   
                 LP LDL   
                 OptiBiotix Health, UK 
               
               
                   
                 TWK-10 
                 Synbio Tech, Taiwan 
               
               
                   
                 GL208 
                 Glac Biotech, Taiwan 
               
               
                   
                 ATCC 14917 T   
                 Bioresource Collection and 
               
               
                   
                   
                 Research Center, Taiwan 
               
               
                   
                   
               
            
           
         
       
     
     The seven different  Lactobacillus plantarum  strains obtained were subjected to amplification with the PCR reagents and conditions shown in Tables 1 and 2 above using the pair of primers having sequences of SEQ ID NOs: 1 and 2, as described in Example 1 above. As shown in  FIG. 2 , none of these seven strains showed amplification product with size around 900 bp. The result showed that the primers are strain-specific for PS128 identification. 
     As a comparison, the primer pair reported by Song et al. (Song YL, Kato N, Liu CX, Matsumiya Y, Kato H, Watanabe K. (2000) “Rapid identification of 11 human intestinal  Lactobacillus  species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA.” FEMS Microbiol. Lett. 187:167-173) with a forward primer having a sequence of 5′-ATTCATAGTCTAGTTGGAGGT-3′(SEQ ID NO: 3) and a reverse primer having a sequence of 5′-CCTGAACTGAGAGAATTTGA-3′(SEQ ID NO: 4) claims to identify human intestinal species  Lactobacillus plantarum  by PCR. 
     The PCR is repeated under the same condition as above with the seven different  Lactobacillus plantarum  strains and PS128, using the primer pair reported by Song et al. The result was shown in  FIG. 3 . The primer pair reported by Song et al. produced similar bands with a PCR product size of 248 bp for all tested species of  Lactobacillus plantarum  including eight strains in addition to the species  Lactobacillus rhamnosus  bacteria. 
     While some of the embodiments of the present disclosure have been described in detail in the above, it is, however, possible for those of ordinary skill in the art to make various modifications and changes to the particular embodiments shown without substantially departing from the teaching and advantages of the present disclosure. Such modifications and changes are encompassed in the spirit and scope of the present disclosure as set forth in the appended claims.