The coefficients of variation (CV) within each duplex 5 nuclease assay were
determined by testing DNA isolated from feces spiked with a monoculture. This
was performed 10 times for determination of the reproducibility and three times
in quadruplicate for repeatability.
Data analyses. For statistical analysis, the software package SPSS for Windows
(version 12.0.1; SPSS, Inc., Chicago, Ill.) was used. All values were checked for
normality by visual inspection of the normal probability plots. Differences were
tested with paired sample t tests, and if P was 0.05 the difference was considered
statistically significant. Although the breast-fed group is compared to the
formula groups, it has to be kept in mind that no complete randomization was
obtained because it is not possible to double blindly assign infants to a breast-fed
group.
TABLE 2. Primers and probes used in the duplex 5 nuclease assays
Target Primers and
probes Sequence (533) Tm
(°C)
%
GC BLAST ID number Amplicon
length (bp)
L. acidophilus F_acid_IS GAA AGA GCC CAA ACC AAG TGA TT 59 43 1089017502-26171-202965955840 85
R_acid_IS CTT CCC AGA TAA TTC AAC TAT CGC TTA 59 37 1089017571-27139-52545094772
P_acid_IS TAC CAC TTT GCA GTC CTA CA 70 45 1089017717-29310-154296055415
L. casei F_case_IS CTA TAA GTA AGC TTT GAT CCG GAG ATT T 59 36 1037022798-023495-2136 132
R_case_IS CTT CCT GCG GGT ACT GAG ATG T 59 55 1037022917-024843-29627
P_case_IS ACA AGC TAT GAA TTC ACT TGC 70 38 1037022752-023005-20772
L. delbrueckii F_delb_IS CAC TTG TAC GTT GAA AAC TGA ATA TCT TAAa 58 30 1089018504-4206-64529811906 94
R_delb_IS CGA ACT CTC TCG GTC GCT TT 58 55 1089018475-6841-166657768151
P_delb_IS CCG AGA ATC ATT GAG ATC 68 44 1089018437-6309-163988227498
L. fermentum F_ferm_IS AAC CGA GAA CAC CGC GTT AT 58 50 1036676682-09669-23287 88
R_ferm_IS ACT TAA CCT TAC TGA TCG TAG ATC AGT CA 58 38 1036676709-010209-2351
P_ferm_IS TAA TCG CAT ACT CAA CTA A 68 32 1036676736-010547-20717
L. paracasei F_paca_IS ACA TCA GTG TAT TGC TTG TCA GTG AAT AC 60 38 1038306417-016220-23561 80
R_paca_IS CCT GCG GGT ACT GAG ATG TTT C 60 55 1038306445-016796-3050
P_paca_IS TGC CGC CGG CCA G 70 85 1038306524-018375-2626
L. plantarum F_plan_IS TGG ATC ACC TCC TTT CTA AGG AAT 58 42 1038305707-03107-18756 144
R_plan_IS TGT TCT CGG TTT CAT TAT GAA AAA ATAa 58 26 1038305742-04177-12861
P_plan_IS ACA TTC TTC GAA ACT TTG T 68 32 1038305778-04682-12880
L. reuteri F_reut_IS ACC GAG AAC ACC GCG TTA TTT 59 48 1089025339-29395-129280047216 93
R_reut_IS CAT AAC TTA ACC TAA ACA ATC AAA GAT TGT CT 59 28 1089025385-30347-37558232754
P_reut_IS ATC GCT AAC TCA ATT AAT 69 28 1089025413-30287-26112845854
L. rhamnosus F_rham_IS CGG CTG GAT CAC CTC CTT T 59 58 1023708254-09591-2284 97
R_rham_IS GCT TGA GGG TAA TCC CCT CAA 59 52 1023708352-010389-16127
P_rham_IS CCT GCA CAC ACG AAA 69 55 1023708453-011313-6655
Lactobacillus spp. F_alllact_IS TGG ATG CCT TGG CAC TAG GA 58 55 1024485925-024664-30598 92
R_alllact_IS AAA TCT CCG GAT CAA AGC TTA CTT AT 58 35 1024478788-024701-16287
P_alllact_IS TAT TAG TTC CGT CCT TCA TC 68 40 1024478009-017753-28422
All bacteria F_eub TCC TAC GGG AGG CAG CAG T 59 –b 466a
R_eub GGA CTA CCA GGG TAT CTA ATC CTG TT 58
P_eub CGT ATT ACC GCG GCT GCT GGC AC 70
a Concessions to the probe and primer design had to be made in these cases (more than three consecutive nucleotides are the same or an amplicon length greater
than 150 bp).
b Nadkami et al.
TABLE 3. Optimized primer and probe concentrations
for the duplex 5 nuclease assays
Target 5 Nuclease
assay
Concn (nM)
Forward
primer
Reverse
primer Probe
L. acidophilus L. acidophilus 900 900 200
All lactobacilli 900 900 200
L. casei L. casei 900 900 200
All lactobacilli 300 300 50
L. delbrueckii L. delbrueckii 300 300 100
All lactobacilli 900 900 200
L. fermentum L. fermentum 300 300 100
All lactobacilli 300 300 100
L. paracasei L. paracasei 300 300 100
All lactobacilli 300 300 100
L. plantarum L. plantarum 300 300 100
All lactobacilli 300 300 100
L. reuteri L. reuteri 300 300 100
All lactobacilli 900 900 200
L. rhamnosus L. rhamnosus 900 450 200
All lactobacilli 150 100 100
Genus Lactobacillus All lactobacilli 600 600 100
All bacteria 300 300 100
VOL. 72, 2006 FECAL LACTOBACILLI IN INFANTS RECEIVING PREBIOTICS 2361
Downloaded from http://aem.asm.org/ on January 17, 2015 by guest