Multiplex PCR detection of waterborne intestinal protozoa: microsporidia, Cyclospora, and Cryptosporidium

Abstract

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 10(1) to 10(2) oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.

Keywords: Cryptosporidium; Cyclospora; microsporidia; multiplex PCR; waterborne protozoa.

Fig. 1

Sensitivity of nested PCR primer sets for microsporidia (A), Cyclospora cayetanensis (B), and Cryptosporidium (C). M, DNA marker; N, negative control (DW).

Fig. 2

Sensitivity of multiplex nested PCR for microsporidia, Cyclospora cayetanensis, and Cryptosporidium. Serially diluted template DNA from each type of protozoan was mixed, and 3 kinds of primer sets for each type were included in 1 reaction tube. M, DNA marker; N, negative control (DW).

Fig. 3

Restriction enzyme digestion of nested PCR products of microsporidia digested with BsaBI (A) and Cryptosporidium parvum digested with BsiEI (B). M, DNA marker; Ei, E. intestinalis; Cp, C. parvum.

Fig. 4

Specificity of multiplex nested PCR primers. No cross-reacted PCR bands were detected when the 3 primer sets were paired with DNA from the different protozoa. Lane 4, negative control (DW).