Pengfei Du

Publications (5)11.54 Total impact

• Article: Rapid identification and differentiation of Theileria sergenti and Theileria sinensis using a loop-mediated isothermal amplification (LAMP) assay.

  Aihong Liu, Guiquan Guan, Pengfei Du, Huitian Gou, Jun Zhang, Zhijie Liu, Milin Ma, Qiaoyun Ren, Junlong Liu, Jifei Yang, Youquan Li, Qinli Niu, Qi Bai, Hong Yin, Jianxun Luo
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  ABSTRACT: The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of two benign bovine Theileria species -T. sergenti and T. sinensis. The LAMP assay is inexpensive and easy to perform and involves a rapid reaction-the amplification can be performed in 55min or 50min under isothermal conditions of 61°C or 63°C, respectively, by employing a set of four species-specific primer mixtures. The results can be checked using agarose gels. The optimal assay conditions, under which the assay exhibited with no cross-reaction with other closely related tick-borne parasites (T. annulata, Babesia bovis, B. bigemina, B. major, B. ovata, B. U. sp., Anaplasma marginale) or between the two Theileria species of interest, was established. The assay is approximately 10-fold more sensitive than the conventional specific PCR assay. The LAMP assay was validated using DNA from 6 standard stocks in the laboratory and was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infection cattle or yaks in China. These findings indicate that this Theileria species-specific LAMP assay may have potential clinical applications for the detection and differentiation of two benign bovine Theileria species -T. sergenti and T. sinensis, especially in endemic countries.
  Veterinary Parasitology 08/2012; · 2.58 Impact Factor
• Article: Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina.

  Aihong Liu, Guiquan Guan, Pengfei Du, Huitian Gou, Zhijie Liu, Junlong Liu, Milin Ma, Jifei Yang, Youquan Li, Qinli Niu, Qiaoyun Ren, Qi Bai, Hong Yin, Jianxun Luo
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  ABSTRACT: Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and/or Babesia bigemina and is characterized by significant morbidity and mortality worldwide. This disease is widespread in most parts of China. However, it is difficult to rapidly discriminate between the B. bovis and B. bigemina species. To detect and distinguish these species, a loop-mediated isothermal amplification (LAMP) platform that targets specific sequences of the internal transcribed spacer (ITS) genes was developed. Specificity testing revealed that there was no cross-reaction with the other tick-borne parasites B. ovate, B. major, unnamed bovine Babesia, Theileria annulata, Theileria sinensis, Theileria sergenti, and Anaplasma marginale, or with bovine white blood cells. The sensitivity of the LAMP method was 0.1pg DNA for both B. bovis and B. bigemina, which was superior to that of the classical PCR methods. This assay was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infected cattle in China. These findings indicate that the Babesia species-specific LAMP assay may have potential clinical application in the detection and differentiation of Babesia species, particularly in countries in which babesiosis is endemic.
  Parasitology International 07/2012; 61(4):658-63. · 2.13 Impact Factor
• Article: A recently identified ovine Babesia in China: Serology and sero-epidemiology.

  Guiquan Guan, Miling Ma, Aihong Liu, Qiaoyun Ren, Jinming Wang, Jifei Yang, Anyan Li, Zhijie Liu, Pengfei Du, Youquan Li, Qing Liu, Hai Zhu, Hong Yin, Jianxun Luo
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  ABSTRACT: Babesia sp. in Xinjiang, transmitted by Hyalomma, is a large Babesia that is infective for small ruminants, but it has almost no pathogenicity in healthy sheep. On the basis of the sequences of the 18S rRNA and internal transcribed spacer (ITS) genes, morphological characteristics, vector tick species and pathogenicity it was identified recently as a novel Babesia species. In the present study, an enzyme-linked immunosorbent assay (ELISA) was developed using soluble merozoite antigens of Babesia sp. in Xinjiang (BXJMA) derived from in vitro culture. When the positive threshold was chosen as 24.65% of the specific mean antibody rate, the specificity and sensitivity were both 97.3%. There was no cross-reaction between BXJMA and positive sera from sheep infected with other Chinese ovine piroplasms or Anaplasma ovis in the ELISA and western blotting. Specific antibodies against Babesia sp. in Xinjiang could be detected 2weeks post infection and a high level of antibodies persisted for more than 12weeks in experimentally infected sheep. The ELISA was tested on 3857 sera collected from small ruminants in 50 prefectures of 22 provinces to evaluate the sero-epidemiology of Babesia sp. in Xinjiang infection, and the average positive rate was 31.66%. These data provide that the developed ELISA is a powerful tool for the sero-diagnosis of Babesia sp. in Xinjiang and confirm that it is a novel species.
  Parasitology International 05/2012; 61(4):532-7. · 2.13 Impact Factor
• Article: Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences.

  Aihong Liu, Guiquan Guan, Pengfei Du, Zhijie Liu, Huitian Gou, Junlong Liu, Jifei Yang, Youquan Li, Milin Ma, Qinli Niu, Qiaoyun Ren, Qi Bai, Hong Yin, Jianxun Luo
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  ABSTRACT: We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata, an economically important cattle disease in China that occurs in subtropical and tropical areas. These assays target the ribosomal RNA (18S rRNA) and ITS LAMP sequences. The primer set for each gene target consists of four primers, and each set recognizes six distinct regions on the target gene to allow for the highly specific detection of T. annulata. The specific ladder bands were amplified from the autologous genomic DNA of four Chinese-laboratory-preserved standard T. annulata stocks, and there were no cross-reactions with the genomic DNA of normal bovine blood and other protozoan species. The LAMP assays were sufficiently sensitive to detect 0.1 pg/μl of genomic DNA. Furthermore, DNA extracted from blood collected from cattle experimentally infected with T. annulata (18-105 days post-infection) was amplified, demonstrating the high sensitivity of these primers. Of the 351 field samples collected from China, 24.5% were positively detected by two LAMP primers, and 18.2% were found to be positive for T. annulata infection by PCR. These results indicate that the LAMP assay could be a potential diagnostic tool for epidemiological studies of T. annulata infection in China.
  Experimental Parasitology 02/2012; 131(1):125-9. · 2.12 Impact Factor
• Article: Continuous in vitro cultivation of a recently identified Babesia that infects small ruminants in China.

  Guiquan Guan, Miling Ma, Aihong Liu, Pengfei Du, Qiaoyun Ren, Youquan Li, Jinming Wang, Zhijie Liu, Hong Yin, Jianxun Luo
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  ABSTRACT: Babesia sp. Xinjiang was isolated from a splenectomised sheep infested by Rhipicephalus sanguineus and Hylomma anatolicum anatolicum, collected from sheep and cattle in Xinjiang province. It was considered to be a novel ovine Babesia species on the basis of its morphology, pathogenicity, vector tick species and alignments of 18S ribosomal RNA (18S rRNA) and internal transcribed spacers (ITS) gene sequences. Continuous in vitro cultures of the ovine parasite were established using infected sheep blood. In RPMI 1640 medium with 7.5% sheep red blood cells (RBCs) maintained in an incubator at 37 °C and 5% CO(2), the percentage of parasitized erythrocytes (PPE) peaked at 10% in 24- and 6-well plates. It increased to 20-50% with the same culture medium but with 2.5% RBC in 75 cm(2) flasks. Two clonal lines of Babesia sp. Xinjiang were screened using the limiting dilution method. Growth characteristics of these lines in vitro were measured by a microtiter-based spectrophotometric method and from the PPE. The generation time in sheep erythrocytes was between 15.20 h and 16.27 h. Furthermore, the host range of parasite was identified with in vitro culture and in vivo infection. Erythrocytes of sheep, cattle, sika deer and humans could be invaded into by lines in vitro, but the parasites could not propagate in human erythrocytes. The parasites could not enter erythrocytes from goats in vitro. However, in vivo, only sheep could be infected by lines. Finally, a Babesia sp. Xinjiang-like parasite (which shared 99.5% identity with the original strain of Babesia sp. Xinjiang) was isolated using this in vitro culture system from 1 of 19 sheep blood samples collected from western Gansu province, China.
  Veterinary Parasitology 02/2012; 187(3-4):371-8. · 2.58 Impact Factor