Nested Polymerase Chain Reaction For Early Detection Of Pheumocystis Carinii In Experiental Rat

Object: Using rat model, to detect the sensitivity mtLSU nested PCR and other two methods in detection of Pneumocystis carinii (P. carinii). And compared the detection rate of P. carinii between lung tissue and BALF (bronchoalveolar lavage fluid, BALF) specimen; To identify a higher sensitive nested PCR method in early stage of P.carinii infected. sequenced the mtLSU and ITS1-5.8S rRNA-ITS2 gene of P.carinii and then compared the genetic difference among these two sequences and those come from different hosts published in GenBank, respectively.Method:Infection of P. carinii in rats was established by subcutaneous injection of an immunodepressant, dexamethasone.The study was carried out in 7 goups with 10 rats each, including one uninfected group as a control. Samples of bronchoalveolar lavage fluid (BALF) and lung tissue from the rats were collected and assayed weekly by nested PCR after week 3. The primer for mtLSU and ITS1-5.8S rRNA-ITS2 of P.carinii was used for the nested PCR. At the same time P. carinii were examined under microscope in lung tissue and BALF smears stained with Gomori's methenamine silver (GMS). The amplification was sequenced with double sequencing method; the sequences were aligned to those sequence with different hosts infected with P. carinii published in Genbank using the program of BLAST .The phylogenic trees were built by DNASTAR. All the results were analysed with statistical methods for detecting the significance among the two nested PCR methods and the microscopic examination way.Result P. carinii was observed by the microscopic examination beginning 5 weeks post-infection, and went to the peak at 6-week and 7-week post-infection. A specific marker site of mtLSU of P.carinii from the samples of LT and BALF in the infected rats were amplified by the nested PCR, and it was found that the positive rates were both 100% in 3-week and 4-week post-infection. Then the positive rate were 100% and 50% (5W), 90% and 100% (6 W), 100% and 80% (7 W) ,100% and 87.5%(8 W) , respectively. And a specific marker site of ITS1-5.8S rRNA-ITS2 of P.carinii from the samples of LT and BALF in the infected rats were amplified by the nested PCR, and it was found that the positive rates were 20% and 0(0/10) (3W),70% and 10%(4W),90% and 30%(5W),90% and 80%(6W),100% and 80%(7W),62.5% and 62.5%(8W),respectively. Meanwhile the statistical analysis showed there was significance between the two nested PCR methods in detecting P.carinii in the first 3 to 4 weeks post-infection when no obvious symptom was appeared in the rats. That is, the sensitivity was higher using mtLSU -nested PCR than that of ITS - nested PCR. While in the stage the infected rats have obvious symptom (5-8 week), there was no significance between the two nested PCR methods. No significant difference also showed on the sensitivity detecting P.carinii using mtLSU nested PCR with lung tissue and BALF specimens. While using ITS - nested PCR the sensitivity detecting in lung tissue was higher than that in BALF. The lengths of mtLSU sequences of P. carinii from Wistar rats were 155bp. The homologies of mtLSU gene of P.carinii among Wistar rats, Wakefieldiae(PCU20170), human from spain (DQ473446 ) ,Pneumocystis murina ( AF257179 ) , Pneumocystis carinii from Macaca fascicularis(AY265389)and human from India were 98.7%,98.7%,87.2%,76.6 and 81.2%. The lengths of ITS1-5.8S rRNA-ITS2 sequences of P.carinii from Wistar rats were 520bp. The homologies of ITS1-5.8S rRNA-ITS2 gene of P.carinii among Wistar rats, Pneumocystis gerbil(AY875972), Pneumocystis rabbit(DQ010098), Pneumocystis Wakefieldiae(L27658), Pneumocystis human(PCU07211)and Pneumocystis murina(AY532651) were72.4%,64.7%,83.1%,63.9% and 75.7%. We found that the 5.8S rDNA gene is conservative,both ITS1 an d ITS2 gene are variant,which indicates that ITS gene is suitablefor the gene variation and phylogenetic evolution study.Conclusions The nested PCR to identify specific marker site of mtLSU is is the most sensitive one in three methods in different stage rat of P.carinii infected, especially in early stage. The identification rate of P.carinii in the BALF sample is equal to that of lung tissues, which indicates it can be applied to the identification of P.carinii in the noninvasive respiratory tract samples in the early stage .We found the mtLSU gene sequence is conservative and only small defference exist in different host of p.carinii. which indicates it is suitable for diagnosis of p.carinii, And we also found 5.8S rRNA gene is highly conservative in which both ITS1 and ITS2 gene are variant,which suggests that ITS gene is suitable for the gene variation and phylogenetic evolution study.