Researches On PCR, LAMP Method And IL-17,-23 And -12 Levels For Detection Of Pneumocystis Carinii Infection In Rats

Pneumocystis carinii (P.carinii) is one of opportunistic pathogenicfungi and occurs mainly in immunocompromised patients such as acquiredimmunodeficiency syndrome (AIDS), malignancy tumor, organtransplantation and chronic obstructive pulmonary disease (COPD). Insevere cases, it can lead to death. Therefore, early accurate diagnosis andtreatment in time is very important for the P. carinii infection. In recentyears, it has been documented that Th17 cells are closely related toinfectious diseases and IL-17 secreted from these cells possesses a certainanti-fungus activity. Besides, IL-12 and IL-23 can promote the productionof IL-17. Nevertheless, the role of Th17 cells and their related cytokines inthe immune response remains to be clearly elucidated. Based on asuccessfully established rat model of P. carinii infection, the experimentalstudies were carried out in two aspects. The fist is to explore diagnosticmethods for P. carinii infection by the loop-mediated isothermalamplification technique (LAMP). The second is to assess the levelvariances of IL-17, IL-23 and IL-12 in bronchialveolar lavage fluid(BALF), supernatant of lung homogenization and culture medium ofalveolar macrophage in vitro.The P. carinii infection animal model was established in thirty femaleWistar rats. After 14 weeks P. carinii infection, rats were anesthetized andperformed endotracheal intubation for collection of BALF. The BALF wascentrifuged, precipitated, suspended and then cultured in a 24-well plate at37℃, 5% CO2 for 24 h. The cell culture medium was harvested for furtherassay. Lung imprint smears were made to detect p. carinii by routineconventional Giemsa staining, and the remains tissues were stored at -80 ℃. The results showed that the cysts of p. carinii could be detected in lungimprint smears derived from infected rats, and proved that the animalmodel had been successfully established, and that the infectious incidenceis 75%. 0.2g frozen lung tissues were homogenized and the supernatantwas collected for interleukin detection and the sediments were carried outto extract DNA using lysate buffer and phenol-chloroform method.Recombinant plasmid pGEX-6p2 carrying nuclear ribosomal small subunit18s rDNA, pGEX-6p2-18s rDNA, and mitochondrial ribosomal largesubunit, pGEX-6p2-mtLSU rDNA, were constructed. The positive cloneswere screened and sequenced. The results showed that the sequences ofthe recombinant plasmids pGEX-6p2-18s rDNA and pGEX-6p2-mtLSUrDNA were consistent with the sequences from Genbank. The LAMPprimers of 18s rDNA and mtLSU rDNA were designed respectively bysoftware online (primerExplorerV4). The genome in rat lungs served astemplate, both of primers were amplified at 63℃for 1h separately. Theproduction were digested by restriction enzyme and the results wereobserved. Moreover, Combinant plasmids were used to detect thesensitivity of LAMPby the copy number of 10-fold serial dilution method.The researches indicated that the ladder strips could be amplified by onlythe LAMP primers of 18s rDNA and the size of the digested fragment ofLAMP products was consistent with the theoretical analysis well.However, the sensitivity of LAMP was not stable, perhaps was due to thepolymorphism of the target gene. The extracted DNA samples from lungswere detected by PCR and nested PCR respectively to determine theinfection rate. The results showed that the nest-PCR was more sensitivethan general PCR and etiological method, but lower specificity thanetiological method and having a certain false-positive. Based on the P.carinii infection model, the concentration of IL-17, IL-23 and IL-12 inBALF, lung homogenization supernatant and culture medium of alveolarmacrophage was measured by ELISA kits, and compared with the control group. The results documented that the concentration of IL -17, IL-23 andIL-12 in BALF of infected rats in experimental group were higher thanthat in BALF in control group respectively, while there were no significantdifferences between uninfected rats in experimental group and the controlgroup.In conclusion, the animal model of P. carinii infection has beensuccessfully established and the infection rate was determined usingetiological, PCR and nest-PCR methods. Moreover, the combinantplasmids pGEX-6p2 carrying 18s rDNA and pGEX-6p2 carrying mtLSUrDNA have been constructed by the genetic recombination. The detectiontechnique of P. carinii by LAMP method is primarily established. Thismethod is simple and quick. However, because of its less stable, it still ispending further study. The concentration of cytokines were assayed byELISAin three samples,and the results suggested that the concentration ofIL-17, IL-23 and IL-12 in BALF of infected rats in experimental groupwere significantly increased, which indicated that IL-17 played importantroles in protecting against fungi infection and strengthening immuneresponds. This paper has discussed the detection methods about P.carinii and immune responds, and also provided a new insight for thepathogenesis of P. carinii infection.