Background
Methods
Patient selection
CSF samples
Bacterial isolates
Species | Strain ID number | RT-PCR | mPCR/RLB | Clinical isolates (No) | ||||
---|---|---|---|---|---|---|---|---|
Target gene | Detection limit | Genome copies/μL | Target gene | Detection limit | Genome copies/μL | |||
E. coli
| ATCC 25922 | 16S rRNA | 2 fg | 0.3 | 16S rRNA | 500 fg | 90 | 6 |
S. aureus
| ATCC 25923 |
femA
| 200 fg | 63 |
nuc
| 500 fg | 160 | 9 |
L. monocytogenes
| ATCC 19112 |
hly
| 200 fg | 62 |
hly
| 50 fg | 15 | 2 |
N. meningitides
| ATCC 29019 |
ctrA
| 20 fg | 8 |
ctrA
| 500 fg | 200 | 2 |
S. pneumoniae | SSI serotype 14 |
lytA
| 200 fg | 90 |
ply
| 500 fg | 230 | 7 |
H. influenza
| ATCC 10211 |
bexA
| 20 fg | 10 |
gyrB
| 500 fg | 255 | 10 |
S. agalactiae
| ATCC A2 |
cfb
| 200 fg | 91 |
cfb
| 500 fg | 228 | 5 |
DNA extraction
RT-PCR
Primer and probe design
Specificity | Target | Primer and probe sequence (5′- 3′) | Application product (bp) | |
---|---|---|---|---|
E.coli
| 16S rRNA | F | GGGAGTAAAGTTAATACCTTTGC | 204 |
R | CTCAAGCTTGCCAGTATCAG | |||
Probe | FAM-CGCGATCACTCCGTGCCAGCAGCCGCGGATCGCG-BHQ1 | |||
L.monocytogenes
|
hly
| F | CAT GGCACCACCAGC ATCT | 64 |
R | ATC CGCGTGTTTCTTTTCGA | |||
Probe | HEX-CGCCTG CAA GTC CTA AGA CGC CA-BHQ1 | |||
S. aureus
|
femA
| F | TGCTGGTGGTACATCAAA | 97 |
R | ACGGTCAATGCCATGATTTAA | |||
Probe | FAM-ATTTTGCCGGAAGTTATGCAGTGCAATG-BHQ1 | |||
N. meningitidis
|
ctrA
| F | TGTGTTCCGCTATACGCCATT | 114 |
R | GCCATATTCACACGATATACC | |||
Probe | FAM-AACCTTGAGCAA“T”CCATTTATCCTGACGTTCT-SpC6 “T”-BHQ1 | |||
H. influenzae
|
bexA
| F | TGCGGTAGTGTTAGAAAATGGTATTATG | 116 |
R | GGACAAACATCACAAGCGGTTA | |||
Probe | HEX-ACAAAGCGTATCAA“T”ACTACAACGAGACGCAAAAA-SpC6 “T”-BHQ | |||
S. pneumoniae
|
lytA
| F | ACGCAATCTAGCAGATGAAGCA | 75 |
R | TCGTGCGTTTTAATTCCAGCT | |||
Probe | FAM-TGCCGAAAACGCTTGATACAGGGAG-BHQ1 | |||
S. agalactiae
|
cfb
| F | CGCAATGAAGTCTTTAATTTTTC | 260 |
R | ATGATGTATCTATCTGGAACTCTAGTG |
Specificity | Thermal profiles |
---|---|
N. meningitides
| 95°C for 2 min, followed by 50 cycles of 95°C for 5 sec and 60°C for 20 sec |
S. aureus
| |
H. influenzae
| |
S. pneumoniae
| |
L.monocytogenes
| 95°C for 2 min, followed by 50 cycles of 95°C for 5 sec and 63°C for 20 sec |
E.coli
| 95°C for 30 sec, followed by 50 cycles of 95°C for 15 sec, 50°C for 30 sec and 75°C 20s |
S. agalactiae
| 95°C for 30 sec, followed by 35 cycles of 94°C for 10 sec, 60°C for 15 sec and 72°C for 25s |
Analytical sensitivity and specificity
mPCR/RLB
Primer and probe design
mPCR/RLB
Analytical specificity and sensitivity for mPCR/RLB
Statistical analysis
Results
General clinical characteristics
Gestational age (week) | |
---|---|
<37 | 5 (9%) |
37-42 | 51 (91%) |
Gender
| |
Male | 36 (64%) |
Female | 20 (36%) |
Onset
| |
Early (<1 week) | 15 (27) |
Late (>1 week) | 41 (73) |
Antibiotics before hospitalization
| |
Yes | 36 (64%) |
No | 20 (36%) |
Syptoms
| |
fever | 51 (91%) |
Jaundice | 23 (41%) |
convulsion | 10 (18%) |
Signs
| |
Bulging fontanelle | 21 (38%) |
Limb muscle tension change | 14 (25%) |
Characteristics | All patients (n = 56) |
---|---|
Blood
| |
Peripheral white cell count
| |
(×109/l) | 22 (39%) |
0- | 34 (61%) |
15- | |
Serum C-reaction protein (mg/l)
| |
0-8 | 35 (63%) |
>8 | 21 (38%)) |
CSF
| |
WBC count ( ×10
6
/L )
| |
0- | 10 (18%) |
21- | 18 (32%) |
>100 | 28 (50%) |
Glucose (mmol/l)
| |
0-2.2 | 34 (61%) |
>2.2 | 22 (39%) |
Protein (mg/l)
| |
204-1000 | 26 (46%) |
1001-2000 | 21 (38%) |
>2000 | 9 (16%) |
CSF culture positive
| 5 (9%) |
Clinical microbiology
Primers and probes
Specificity | Primer/probe | Target |
Tm (°C) | GenBank accession No. | Primer and probe sequence (5′-3′) | Application product (bp) |
---|---|---|---|---|---|---|
S. aureus
| SanucSb |
nuc
| 65.68 | V01281 | GCG ATT GAT GGT GAT ACG GTT | 278 |
S. aureus
| SanucAb |
nuc
| 69.12 | V01281 | AGC CAA GCC TTG ACG AAC TAA AGC | |
S. aureus
| SanucSp |
nuc
| 61.06 | V01281 | GAT GGA AAA ATG GTA AAC GAA G | |
S. aureus
| SanucAp |
nuc
| 61.36 | V01281 | CAT TGG TTG ACC TTT GTA CAT TAA | |
S. pneumoniae
| SpplySb |
ply
| 67.47 | M17717 | CCC ACT CTT CTT GCG GTT GA | 208 |
S. pneumoniae
| SpplyAb |
ply
| 61.68 | M17717 | TGA GCC GTT ATT TTT TCA TAC TG | |
S. pneumoniae
| SpplySp |
ply
| 65.44 | M17717 | CCC AGC AAT TCA AGT GTT CG | |
S. pneumoniae
| SpplyAp |
ply
| 65.49 | M17717 | CCA CTT GGA GAA AGC TAT CGC T | |
L.monocytogenes
| LmhlySb |
hly
| 67.37 | M24199 | CAT GGC ACC ACC AGC ATC T | 135 |
L.monocytogenes
| LmhlyAb |
hly
| 63.8 | M24199 | CAC TGC ATC TCC GTG GTA TAC TAA | |
L.monocytogenes
| LmhlySp |
hly
| 68.2 | M24199 | GAA AAG AAA CAC GCG GAT GAA ATC | |
L.monocytogenes
| LmhlyAp |
hly
| 65.33 | M24199 | TGG CGT CTT AGG ACT TGC AG | |
S. agalactiae
| GBScfbSb |
cfb
| 59.53 | X72754 | ATG ATG TAT CTA TCT GGA ACT CTA GTG | 259 |
S. agalactiae
| GBScfbAb |
cfb
| 60.48 | X72754 | CGC AAT GAA GTC TTT AAT TTT TC | |
S. agalactiae
| GBScfbSp |
cfb
| 59.74 | X72754 | ATC AAA GAT AAT GTT CAG GGA AC | |
S. agalactiae
| GBScfbAp |
cfb
| 58.55 | X72754 | TAC TTC TAA TAC AGC TGG TGA AAA | |
N. meningitidis
| NmctrASb |
ctrA
| 66.14 | AF520909 | GCT GCG GTA GGT GGT TCA A | 110 |
N. meningitidis
| NmctrAAb |
ctrA
| 66.36 | AF5209 | TTG TCG CGG ATT TGC AAC TA | |
N. meningitidis
| NmctrASp |
ctrA
| 64.1 | AF5209 | ACG AAC TGT TGC CTT GGA AG | |
N. meningitidis
| NmctrAAp |
ctrA
| 63.76 | AF5209 | ATT GCC ACG TGT CAG CTG | |
H. influenzae
| HigyrBSb |
gyrB
| 62.57 | U32738 | GAA GCA CAG TCA TAA TAA CTT CTG CT | 233 |
H. influenzae
| HigyrBAb |
gyrB
| 63.68 | U32738 | AGC GTC CTG GTA TGT ATA TCG G | |
H. influenzae
| HigyrBSp |
gyrB
| 62.96 | U32738 | TTG CAC CGA TAC AGA ATT ATC ATC | |
H. influenzae
| HigyrBAp |
gyrB
| 63.57 | U32738 | CGG GAT TCC TGT GGA TAT TC | |
E.coli
| Ecoli16SSb | 16SrRNA | 65.74 | J01859 | ATG CCG CGT GTA TCA AGA A | 93 |
E.coli
| Ecoli16SAb | 16SrRNA | 68.03 | J01859 | TAA CGT CAA TGA GCA A | |
E.coli
| Ecoli16SSp | 16SrRNA | 65.93 | J01859 | GGG GAG GAA GGG AGT AAA GT | |
E.coli
| Ecoli16SAp | 16SrRNA | 63.71 | J01859 | AGT ACT TTA CAA CCC GAA GGC |
Results of the RT-PCR and mPCR/RLB analysis
Detection of potential pathogens in CSF by RT-PCR and mPCR/RLB assays
Comparison of RT-PCR assay with mPCR/RLB assay and CSF bacterial cultures
RT-PCR (N = 56) | Total | |||
---|---|---|---|---|
+ | - | |||
mPCR/RLB | + | 14 (25.0%) | 2 (3.6%) | 16 (28.6%) |
- | 11 (19.6%) | 29 (51.8%) | 40 (71.4%) | |
Total | 25 (44.6%) | 31 (55.4%) | 56 |
Cultures(N = 56) | Total | |||
---|---|---|---|---|
+ | - | |||
RT-PCR | + | 2 (3.5%) | 23 (41.1%) | 25(44.6%) |
- | 3 (5.4%) | 28 (50.0%) | 31 (55.4%) | |
Total | 5(8.9%) | 51(91.1%) | 56 |
Cultures (N = 56) | Total | |||
---|---|---|---|---|
+ | - | |||
mPCR/RLB | + | 4 (80.0%) | 12 (23.5%) | 16 (28.6%) |
- | 1 (20.0%) | 39 (76.5%) | 40 (71.4%) | |
Total | 5 (8.9%) | 51 (91.1%) | 56 |