CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES ›› 2023, Vol. 41 ›› Issue (1): 36-43.doi: 10.12140/j.issn.1000-7423.2023.01.006
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LI Mei*(), XIAO Ning, XIA Zhigui
Received:
2022-03-29
Revised:
2022-06-06
Online:
2023-02-28
Published:
2023-02-26
Contact:
* E-mail: CLC Number:
LI Mei, XIAO Ning, XIA Zhigui. Establishment and application of a specific primers-based qPCR targeting asexual stage 18S rDNA for detecting five Plasmodium species[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(1): 36-43.
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URL: https://www.jsczz.cn/EN/10.12140/j.issn.1000-7423.2023.01.006
Table 1
Localization of conservative region of A-type 18S rDNA of P. falciparum and homology comparison with 18S rDNA of other species
保守区编号 Code of conservative area | 在Pf-A上的位置 Location on Pf-A | 片段长度/bp Fragment length/bp | 一致性/% Identification/% | ||
---|---|---|---|---|---|
Po-S | Pf-S | 其他10条 Other 10 sequences | |||
C1 | 1~65 | 65 | 95 | 98 | 100 |
C2 | 85~126 | 42 | 98 | 98 | 98 |
C3 | 298~480 | 183 | 96 | 98 | ≥ 99 |
C4 | 550~644 | 95 | 99 | 98 | 100 |
C5 | 942~1 100 | 159 | 93 | 94 | ≥ 97 |
C6 | 1 184~1 454 | 271 | 98 | 99 | 100 |
C7 | 1 638~1 724 | 87 | 92 | 97 | 100 |
C8 | 1 831~1 978 | 148 | 92 | 97 | ≥ 98 |
C9 | 2 003~2 092 | 90 | 92 | 97 | 100 |
Table 2
Primer sequences sreened for detection 5 species of Plasmodium and their characteristics
引物名称 Primer name | 引物序列(5´→3´) Primer sequence (5´→3´) | 在18S rDNA上的位置 Location on 18S rDNA | 片段长度/bp Fragment length/bp | Tm值a/℃ Tm valuea /℃ | 扩增效率/% Amplification efficiency/% | |
---|---|---|---|---|---|---|
疟原虫属 Plasmodium spp. | Pg-F | GCGCAAGCGAGAAAGTTAAAAG | C6 | 145 | 82 | 98.8 |
Pg-R | CCGCTAATTAGCAGGTTAAGAT | C6 | ||||
Pf | Pf-F | TTATTATCCTTTGATTTTTATCTTTGG | V2 | 108 | 72 | 90.6 |
Pf-R | ATAAATTTATTACGTGTTACTTCTTTG | V2 | ||||
Pv[ | Pv-F | TTTTAAGGACTTTCTTTGCTTCGG | V3 | 321 | 79.5 ~ 80.0 | 92.7 |
Pv-R | CGACGGTATCTGATCGTCTTC | C4 | ||||
Pm | Pm-F | CAAGCGTTATATTTTTTCTGTTACAT | V3 | 165 | 73.5 ~ 74.5 | 96.0 |
Pm-R | GAAACTGTTTTAACTGTTTGCTTTG | V3 | ||||
Po | Po-F | TATAAGATGCTTAGGCAATACAAC | V3 | 177 | 76.0 ~ 77.5 | 97.3 |
Po-R | TCAAACGCAGTAAAATGCTTTAACT | V3 | ||||
Pk | Pk-F | GAAGAAAATATTGGAATTACGTTAAAT | V5 | 90 | 73.5 ~ 74.0 | 95.7 |
Pk-R | CATTCATACGCAAAAAAGAAATAGAT | V5 |
Table 3
The highest concentration of non-target species DNA when the amplification result of 5 pairs of species-specific primers is negative
引物对 Primer pairs | 非目标DNA的最高浓度/拷贝·μl-1 The highest concentration of non-target DNA/copy·μl-1 | ||||
---|---|---|---|---|---|
Pf | Pv | Pm | Po | Pk | |
Pf-F/Pf-R | 5.0 × 108 | 5.0 × 107 | 5.0 × 107 | 5.0 × 107 | |
Pv-F/Pv-R | 5.0 × 108 | 5.0 × 108 | 5.0 × 108 | 5.0 × 107 | |
Pm-F/Pm-R | 5.0 × 106 | 5.0 × 106 | 5.0 × 106 | 5.0 × 107 | |
Po-F/Po-R | 5.0 × 107 | 5.0 × 106 | 5.0 × 106 | 5.0 × 107 | |
Pk-F/Pk-R | 5.0 × 106 | 5.0 × 106 | 5.0 × 107 | 5.0 × 107 |
Table 4
Results of qPCR amplification of target DNA with the lowest detection limit concentration and mixed DNA of target DNA and non-target species by five pairs of species-specific primers
引物对 Primer pairs | 目标DNA Target DNA | 目标和非目标DNA混合 Mix of target DNA and non-target DNA | |||||||
---|---|---|---|---|---|---|---|---|---|
5.0 × 102拷贝·μl-1 5.0 × 102 copy·μl-1 | 5.0 × 106拷贝·μl-1 5.0 × 106 copy·μl-1 | 5.0 × 102拷贝·μl-1 5.0 × 102 copy·μl-1 | |||||||
平均Ct值 Average Ct values | 变异系数/% Coefficient of variation/% | 平均Ct值 Average Ct values | 变异系数/% Coefficient of variation/% | 非目标DNA浓度/拷贝·μl-1 Concentration of non-target DNA/copy·μl-1 | 平均Ct值 Average Ct values | 变异系数/% Coefficient of variation/% | |||
Pf-F/Pf-R | 34.1 ± 0.8 | 2.5 | 19.9 ± 0.0 | 0.1 | 5.0 × 106 | 34.3 ± 0.1 | 0.3 | ||
Pv-F/Pv-R | 30.6 ± 0.5 | 1.5 | 17.5 ± 0.0 | 0.0 | 5.0 × 107 | 31.6 ± 0.9 | 3.0 | ||
Pm-F/Pm-R | 32.1 ± 0.0 | 0.1 | 18.4 ± 0.1 | 0.4 | 5.0 × 106 | 32.6 ± 0.4 | 1.4 | ||
Po-F/Po-R | 31.7 ± 0.7 | 2.1 | 17.6 ± 0.0 | 0.0 | 5.0 × 107 | 31.4 ± 0.4 | 1.2 | ||
Pk-F/Pk-R | 33.6 ± 0.1 | 0.3 | 19.4 ± 0.0 | 0.0 | 5.0 × 106 | 33.4 ± 0.4 | 1.2 |
Table 5
6 Minimum parasite density detected with different PCR with six pairs of genus-/species- specific primers
引物对 Primer pairs | 样品 Sample | 最低检出原虫密度/虫·μl-1 Minimum parasite density/Parasite·μl-1 | ||
---|---|---|---|---|
一步反转录qPCR One-step reverse transcription qPCR | qPCR | 巢式PCR Nest PCR | ||
Pg-F/Pg-R | X差值a最大的恶性疟原虫样品 P. falciparum sample with maximum D-value of Xa | 0.08 | 2.00 | |
Pf-F/Pf-R | X差值最大的恶性疟原虫样品 P. falciparum sample with maximum D-value of X | 0.02 | 2.00 | 9.9 |
X差值最小的恶性疟原虫样品 P. falciparum sample with mimimum D-value of X | 0.40 | 0.40 | 2.0 | |
Pv-F/Pv-R | X差值最大的间日疟原虫样品 P. vivax sample with maximum D-value of X | 0.01 | 0.30 | 6.9 |
X差值最小的间日疟原虫样品 P. vivax sample with mimimum D-value of X | 10.80 | 10.80 | 10.8 | |
Pm-F/Pm-R | X差值最大的三日疟原虫样品 P. malariae sample with maximum D-value of X | 0.30 | 7.40 | 7.4 |
X差值最小的三日疟原虫样品 P. malariae sample with mimimum D-value of X | 10.6 | 10.60 | 10.6 | |
Po-F/Po-R | X差值最大的卵形疟原虫样品 P. ovale sample with maximum D-value of X | 0.06 | 7.70 | 7.7 |
X差值最小的卵形疟原虫样品 P. ovale sample with mimimum D-value of X | 10.00 | 10.00 | 10.0 | |
Pk-F/Pk-R | 诺氏疟原虫样品 P. knowlesi sample | 8.30 | 8.30 | |
平均最低检出限 Everage detection limit | 4.1 ± 5.1 | 6.0 ± 4.3 | 8.2 ± 2.9 |
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