Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA.
Lactobacillus casei strain Shirota (LcS) has been used in the production of fermented milk products for many years and is one of the most intensively studied probiotics. To evaluate the ability of LcS to proliferate in human intestines after it has been ingested, we developed a PCR-based method to identify and quantify LcS using an LcS-specific primer set (pLcS) derived from a randomly amplified polymorphic DNA (RAPD) analysis. We confirmed the high specificity of the pLcS primer set in 167 bacterial strains (57 strains of L. casei and 110 other strains of bacteria commonly isolated from human feces). The method's ability to identify LcS matched that of an ELISA using a monoclonal antibody and a RAPD analysis in a representative sample of colonies cultured from human feces. The detection limit of quantitative PCR (qPCR) using pLcS was 10(4.6) per gram of feces. The number of LcS in feces detected with qPCR was highly and significantly correlated with the number of LcS added to fecal samples within the range of 10(4.6) to 10(9.6) per gram feces (r(2)=0.999, P<0.001). After 14 healthy subjects ingested 10(11.0) CFU of LcS daily for 7 days, 10(9.1+/-0.5) LcS g(-1) (mean+/-S.D.) was detected in the fecal samples of all subjects by qPCR, and 10(8.0+/-0.9) CFU g(-1) was detected by culture; these values were significantly different (P<0.001, paired t-test). After the subjects stopped ingesting LcS, fecal LcS counts obtained with both methods decreased daily. The values produced by the 2 methods might have differed because of an overestimation in the PCR analysis due to the presence of dead LcS cells or an underestimation in the culture system due to the use of selective culture media; however, dead LcS cells can also be beneficial as immunomodulators. We confirmed that qPCR with an LcS-specific primer set was a rapid and accurate method for determining the total amount of LcS in feces including dead or less active cells which could not be detected by culture method.
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan. junji-fujimoto@yakult.co.jp
Junji
J
Fujimoto
Takahiro
T
Matsuki
Masae
M
Sasamoto
Yasuaki
Y
Tomii
Koichi
K
Watanabe
D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D015169:Colony Count, Microbial / Q000379:methods / Q000592:standards; D043302:Cultured Milk Products / Q000382:microbiology; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005243:Feces / Q000382:microbiology*; D005516:Food Microbiology; D006801:Humans; D007780:Lacticaseibacillus casei / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification; D019936:Probiotics; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013997:Time Factors
D000911:Antibodies, Monoclonal; D017931:DNA Primers