Background
Methods
Chemical and reagents
Study area
Selection of subjects
Sampling design
Separation of red blood cells
Parasite DNA isolation
Primer designing and PCR amplification of pvdhfr and pvdhps genes
Gene | Primer name | Primer sequence | Product size (bp) | PCR cycling conditions |
---|---|---|---|---|
Nest I | ||||
pvdhfr
| A1F | 5′ATGGAGGACCTTTCAGATGTATTTGACATT 3′ | 720 | 105 °C for 5 min; (95 °C for 30 s; 50 °C for 30 s; 72 °C for 50 s) × 30 cycles; 72 °C for 10 min |
A1R | 5′GGCGGCCATCTCCATGGTTATTTTATCGTG 3′ | |||
pvdhps
| 2A F | 5′ATTCCAGAGTATAAGCACAGCACATTTGAG3′ | 840 | 104 °C for 5 min; (95 °C for 50 s; 57 °C for 1 min; 72 °C for 1 min) × 35 cycles; 72 °C for 10 min |
2A R | 5′CTAAGGTTGATGTATCCTTGTGAGCACATC 3′ | |||
Nest II | ||||
pvdhfr
| A2F | 5′TTTATGATGGAACAAGACTGGGACGTT 3′ | 701 | 100 °C for 5 min; (95 °C for 45 s; 52 °C for 40 s; 72 °C for 1 min) × 32 cycles; 72 °C for 10 min |
A2R | 5′TCACACGGGTAGGCGCCGTTGATCCTCGTG 3′ | |||
pvdhps
| 2 B F | 5′AATGGCAAGTGATGGGGCGAGCGTGATT 3′ | 610 | 95 °C for 5 min; (94 °C for 40 s; 54 °C for 55 s; 72 °C for 1 min) × 35 cycles; 72 °C for 10 min |
2B R | 5′CAGTCTGCACTCCCCGATGGCCGCGCCAC 3′ |
DNA sequencing of pvdhfr and pvdhps genes
Statistical analysis
Results
Polymorphism of pvdhfr gene
Mutation assessment of pvdhps gene
Regional distribution of pvdhfr and pvdhps haplotype
Variation and distribution of different tandem repeat
Gene | Type | Tandem repeat | KMC (%) | Purulia (%) |
---|---|---|---|---|
Dhfr | Type 1 | GDNTSGGDNTHGGDNTHGGDNADKL | 19.23 | 0.00 |
Type 2 | GDNTSGGDNTHG ------------GDNADKL | 80.77 | 100 | |
Dhps | Type A | GEAKLTNGEGKLTNGEAKLTNGEGKLTNG EAKLTNGEGKLTNGDAKLTNGDSKLTNG | 10.64 | 21.15 |
Type B | GEAKLTNGEGKLTNGEAKLTNGEGKLTNG
-------------------------------------DSKLTNG | 89.36 | 63.47 | |
Type C | GEAKLTNGEGKLTNGEAKLTNGEGKLTNG EAKLTNGEGKLTN
-------------GDSKLTNG | 0.00 | 15.38 |
Combination mutation of pvdhfr and pvdhps gene and emergence of quadruple and quintuple mutant allele
Genotype | Isolates in KMC | Isolates in P | PVDHFR | PVDHPS | Allele type | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
15 | 33 | 50 | 57 | 58 | 61 | 64 | 117 | 173 | 373 | 380 | 382 | 383 | 384 | 512 | 553 | 585 | 601 | ||||
G1 | 11 | 17 | A | P | N | F | S | T | V | S | I | S | E | S | A | P | K | A | V | M | Wild |
G2 | 3 | 1 | A | P | N | F |
R
| T | V | S | I | S | E | S | A | P | K | A | V | M | SM |
G3 | 6 | 6 | A | P | N | F |
R
| T | V |
N
| I | S | E | S | A | P | K | A | V | M | DM |
G4 | 0 | 4 | A | P | N | F | S | T | V |
N
| I | S | E | S | A | P | K | A | V | M | SM |
G5 | 8 | 3 | A | P | N | F | S | T | V | S | I | S | E | S |
G
| P | K | A | V | M | SM |
G6 | 15 | 7 | A | P | N | F |
R
| T | V |
N
| I | S | E | S |
G
| P | K | A | V | M | TM |
G7 | 7 | 0 | A | P | N | F |
R
| T | V |
N
| I | S | E | S |
G
| P | K |
G
| V | M | QM |
G8 | 0 | 6 | A | P | N | F |
R
| T | V | S | I | S | E | S | A | P | K |
G
| V | M | DM |
G9 | 0 | 3 | A | P | N | F | S | T | V | S | I | S | E | S | A |
L
| K | A | V | M | SM |
G10 | 2 | 0 | A | P | N |
L
|
R
| T | V |
N
| I | S | E | S |
G
| P | K |
G
| V | M | QTM |