Background
Herbal medicines
Molecular species identification
Materials and methods
Primers used for HRM analysis
Plant samples and DNA isolation
Species/Type | Abbreviation | Source | Sample type |
---|---|---|---|
Thunbergia laurifolia
| T1 | Materia Medica garden | Fresh |
Faculty of Pharmacy, Chiang Mai University | |||
Thunbergia laurifolia
| T2 | Department of Biology | Fresh |
Faculty of Science, Chiang Mai University | |||
Thunbergia laurifolia
| T3 | Queen Sirikit Botanical Garden | Dry |
Mae Rim, Chiang Mai (voucher number 46323) | |||
Thunbergia laurifolia
| T4 | Queen Sirikit Botanical Garden | Dry |
Mae Rim, Chiang Mai (voucher number 59427) | |||
Crotalaria spectabilis
| C1 | Materia Medica garden | Fresh |
Faculty of Pharmacy, Chiang Mai University | |||
Crotalaria spectabilis
| C2 | Materia Medica garden | Fresh |
Faculty of Pharmacy, Chiang Mai University | |||
Crotalaria spectabilis
| C3 | CMU Biology Garden | Fresh |
Faculty of Science, Chiang Mai University | |||
Commercial CN-C | COM1 | Chiang Mai | Capsule |
Commercial TT-C | COM2 | Chiang Mai | Capsule |
Commercial HBO-C | COM3 | Chiang Mai | Capsule |
Commercial HBO-T | COM4 | Chiang Mai | Tea bag |
Commercial APB-P | COM5 | Lamphun | Powder |
Commercial NK-L | COM6 | Lamphun | Dried leaf |
Commercial GT-L | COM7 | Pa Yao | Dried leaf |
Commercial OTOP-T | COM8 | Pa Yao | Tea bag |
Commercial RTN-S1 | COM9 | Pa Yao | Dried bark |
Commercial RTN-S2 | COM10 | Pa Yao | Dried bark |
Real-time PCR amplification and high resolution melting (HRM) analysis
Primer name | Nucleotide sequence (5’ to 3’) | Ta (°C) | Expected size (bp) |
---|---|---|---|
matK _F | CTTCTTATTTACGATTAACATCTTCT | 57 | 160 |
matK _R | TTTCCTTGATATCGAACATAATG | ||
rbcL_F | GGTACATGGACAACTGTGTGGA | 57 | 150 |
rbcL _R | ACAGAACCTTCTTCAAAAAGGTCTA | ||
rpoC_F | CCSATTGTATGGGAAATACTT | 57 | 170 |
rpoC _R | CTTACAAACTAATGGATGTAA | ||
trnL_F | GAATCGACCGTTCAAGTATCC | 57 | 150 |
trnL _R | TATAGGAAACCCATATTTGATCCAATC |
Authenticating test of herbal products sold on Thai local markets
Results and discussion
Data mining and primers used
Regions |
mat
K
|
rbc
L
|
rpoC
|
trn
L
|
---|---|---|---|---|
Available species | 5 | 6 | 5 | 19 |
Variable characters (%) | 30.67 | 10.07 | 10.19 | 8.90 |
Conserved forward primer/total (%) | 15/26 (57.69) | 21/22 (95.45) | 18/21 (85.71) | 19/21 (90.48) |
Conserved reverse primer/total (%) | 19/23 (82.61) | 25/25 (100) | 19/21 (90.48) | 24/27 (88.89) |
Average %GC content | 35.20 | 46.60 | 44.26 | 34.50 |
Species | Regions | |||
---|---|---|---|---|
trnL
|
matK
|
rbcL
|
rpoC
| |
Crotalaria spectabilis
| HM208335 | AB649973 | - | - |
Thunbergia affinis
| AB817377 | - | - | - |
EU315886 | ||||
Thunbergia alata
| AF061820 | HQ384512 | HQ384878 | - |
EU529130 | AF531811 | |||
EU315887 | ||||
Thunbergia angulata
| EU315888 | - | - | - |
Thunbergia arnhemica
| EU315889 | - | - | - |
Thunbergia atriplicifolia
| EU315890 | - | - | - |
Thunbergia battiscombei
| EU315891 | - | - | - |
Thunbergia capensis
| EU315892 | AM234783 | AM234783 | - |
Thunbergia coccinea
| EU529131 | HG004920 | KF181493 | - |
Thunbergia convolvulifolia
| EU315894 | - | - | - |
Thunbergia dregeana
| EU315895 | - | - | - |
Thunbergia erecta
| AF061821 | AB649972 | - | - |
JQ764614 | ||||
EU529132 | ||||
EU315896 | ||||
Thunbergia fragrans
| U315897 | - | - | - |
Thunbergia galpinii
| EU315898 | - | - | - |
Thunbergia grandiflora
| EU315899 | AB649971 | JQ590086 | - |
JQ586429 | ||||
JQ586428 | ||||
JQ586427 | ||||
Thunbergia gregoryi
| EU315901 | - | - | - |
Thunbergia guerkeana
| EU315901 | - | - | - |
Thunbergia kirkii
| EU315902 | - | - | - |
Thunbergia laurifolia
| - | AB649970 | - | - |
Thunbergia mysorensis
| - | - | AY008828 | - |
Thunbergia petersiana
| EU315904 | - | - | - |
Thunbergia pondoensis
| EU315905 | - | - | - |
Thunbergia togoensis
| EU315906 | - | - | - |
Thunbergia usambarica
| - | - | L12596 | - |
Finding suitable primer pairs for discrimination between T. laurifolia and C. spectabilis
Species | Abbreviation | Tm (oC) | |||
---|---|---|---|---|---|
mat
K
|
rbc
L
|
rpo
C
|
trn
L
| ||
T. laurifolia
| T1 | - | 82.5 ± 0.07 | 80.2 ± 0.12 | 78.8 ± 0.07 |
T. laurifolia
| T2 | - | 82.4 ± 0.14 | 80.1 ± 0.15 | 78.9 ± 0.07 |
T. laurifolia
| T3 | 77.9 ± 0.35 | 82.3 ± 0.14 | 80.2 ± 0.00 | 78.8 ± 0.00 |
T. laurifolia
| T4 | - | 82.4 ± 0.07 | 80.2 ± 0.07 | 78.8 ± 0.14 |
C. spectabilis
| C1 | 75.6 ± 0.21 | 81.6 ± 0.00 | 80.9 ± 0.06 | 78.4 ± 0.14 |
C. spectabilis
| C2 | 75.3 ± 0.07 | 81.6 ± 0.07 | 80.9 ± 0.06 | - |
C. spectabilis
| C3 | 75.4 ± 0.21 | 81.5 ± 0.00 | 80.8 ± 0.00 | 78.2 ± 0.00 |