LincRNA-Cox2 regulates IL6/JAK3/STAT3 and NF-κB P65 pathway activation in Listeria monocytogenes-infected RAW264.7 cells.

Listeria monocytogenes (Lm) can lead to high mortality rates relative to other foodborne pathogens. Lm-induced inflammation is partly characterized by macrophage activation. Long non-coding RNAs (lncRNAs) have important roles in various biological processes. However, it is unknown how lncRNAs regulate the host response to Lm infection. To identify the role of lncRNA in Lm infection, we used in vitro and in vivo models. We found that lincRNA-Cox2 was highly expressed in Lm-infected RAW264.7 cells. LincRNA-Cox2 knockdown resulted in reduced proinflammatory cytokines, apoptosis, migration ability and enhanced phagocytosis of Lm. LincRNA-Cox2 knockdown also reduced the phosphorylation of Janus kinase 3 (JAK3) and signal transducer and activator of transcription (STAT3) and the nuclear translocation of nuclear factor (NF)-κB P65, which are known to be involved in inflammatory responses. Experimentally inhibiting the protein and phosphorylation levels of STAT3 resulted in reduced proinflammatory cytokines and enhanced phagocytosis of Lm by the RAW264.7 cells. Our research suggests that lincRNA-Cox2 plays important roles in inflammation, the phagocytic function and cell migration ability of RAW264.7 cells by activating interleukin (IL)-6/JAK3/STAT3 signaling, and lincRNA-Cox2 also regulates NF-κB P65 nuclear translocation. Our research provides new insights into the regulatory role of lincRNA-Cox2 in Lm infection.

Listeria monocytogenes crosses blood brain barrier through Rho GTPases induced migration of macrophages and inflammatory interleukin expression.

Listeria monocytogenes crossing the blood-brain barrier in the form of "Trojan Horse" is of great significance for the establishment of bacterial encephalitis and meningitis. Induction of cell migration and crossing the blood-brain barrier is very important to understand the Listeria pathogenesis. The Rho GTPases family is considered a key factor in regulating cell migration. This study was designed to investigate the expression of Rho GTPases and their effect on the behavior of cell migration and the stimulation of immune factors. Selective Rho GTPases were investigated by real-time PCR and Western blot. Among these, the expression of RhoA was significantly increased following the infection of Listeria monocytogenes in macrophages. Further, we found that RhoA improves the migration of macrophages and expression of IL-1ß, IL-6, and TNF-α. The expression of IL-1ß, IL-6 and TNF-α possibly facilitates the migration and adhesion of macrophages to cross the blood-brain barrier. This study provides preliminary ground to investigate the detailed mechanism of Listeria monocytogenes crossing the blood-brain barrier.

Induction of FAS II Metabolic Disorders to Cause Delayed Death of Toxoplasma gondii.

The exact mechanism of delayed death of Toxoplasma gondii is not known. FAS II synthesis in the apicoplast of T. gondii is essential for the survival of Toxoplasma gondii, while ß-hydroxyacylacyl carrier protein dehydratase (FabZ) is indispensable for fatty acid synthesis. The present study investigated the relationship between the delayed death of T. gondii by inducing metabolic disorders due to suppression the expression of FabZ. A tetracycline-induced knockout vector inserted with T. gondii fabZ gene was constructed, and transfected into T. gondii TATi strain by electroporation. The stable mutants with tetracycline-induced knockout were selected, expression of FabZ was suppressed by using anhydrotetracycline (ATc), and FAS II deficient tachyzoites were prepared. The Western blot and qPCR results revealed that proliferation of FAS II deficient tachyzoites was not significantly different compared to the normal tachyzoites at 24 h and 48 h; however, after 72 h, the number of T. gondii tachyzoites in the ATc treated group was significantly (p < 0.05) less than that of non-treated group, indicating the delayed death of T. gondii caused by the loss of apicoplast and decrease in the expression of FabZ, which inhibited the FAS II metabolism. The results of this study can be used for prevention of toxoplasmosis by inducing delayed death of T. gondii.

The association between interleukin-19 concentration and diabetic nephropathy.

BACKGROUND: Interleukin-19 (IL-19) is a newly discovered cytokine belonging to the Interleukin-10(IL-10) family. IL-19 have indispensable functions in many inflammatory processes and also can induce the angiogenic potential of endothelial cells. The purpose of present study was to investigate the relation of serum interleukin-19 (IL-19) levels with diabetic nephropathy (DN). METHODS: Two hundred study groups of patients with type 2 diabetes mellitus (T2DM) (109 males and 91 females) were recruited, included normoalbuminuria(n = 102), microalbuminuria(n = 72) and macroalbuminuria(n = 26) . The 50 healthy blood donors were enrolled for the control group. All subjects were assessed for: IL-19, High-sensitivity C-reactive protein (Hs-CRP), Cystatin C, urinary albumin excretion rate (UAE) and glycosylated hemoglobin A1c(HbA1c). RESULTS: The serum IL-19 levels in DN patients were found to be significantly higher compared to controls. IL-19 levels were significantly positively correlated with Hs-CRP, Cystatin C, UAE and HbA1c(r = 0.623, 0.611,0.591 and 0.526 respectively, P < 0.01). Multivariable logistic regression analysis showed IL-19 levels (P = 0.01) were found to be independently associated with patients with DN. CONCLUSIONS: IL-19 is significantly positive correlated with UAE and Cystatin C. IL-19 may play an important role that contributes to the progression of diabetic nephropathy.

Toxoplasma gondii ROP18: potential to manipulate host cell mitochondrial apoptosis.

Toxoplasma gondii is an obligate intracellular parasite that may manipulate host cell mitochondrial apoptosis pathways. In our experiment, 293T cells were transfected with the p3×FLAG-CMV-Myc-ROP18 vector and expressed the ROP18-Myc fusion protein. Cell apoptosis was induced by 0.5 µg/mL actinomycin D (ActD) and was detected by Annexin V-FITC/PI assay. The cell mitochondrial membrane potential was determined by JC-1. Cytochrome c (Cyto-c) from mitochondria and the cytoplasm was measured by Western blot. The Bcl-2 and Bax coding gene expression levels were detected by real-time PCR. We found, in vitro, that T. gondii ROP18 significantly suppressed 293T cell apoptosis induced by ActD and maintained mitochondrial membrane potential and integrity, thereby preventing the release of Cyto-c from mitochondria into the cytoplasm. The ratio of Bcl-2/Bax in ROP18-overexpressing cells was significantly higher than that of the negative control. Therefore, we speculate that ROP18 could suppress host cell apoptosis via the mitochondrial apoptosis pathway in vitro.

Toxoplasma gondii nucleus coding apicoplast protein ACP synthesis and trafficking in delayed death.

This study aimed to explore Toxoplasma gondii nucleus coding apicoplast protein acyl carrier protein (ACP) synthesis and trafficking in delayed death. The recombinant T. gondii ACP was expressed by prokaryotic expression method, and anti-ACP polyclonal antibody was obtained from rabbit immune. T. gondii "delayed death" was induced by clindamycin (CLDM), and ACP transcription was determined by real-time PCR assay. The expression of ACP with transit type (t-ACP) and mature type (m-ACP) was determined by Western blotting with anti-ACP polyclonal antibody. The mutant-expressed ACP fused with green fluorescent protein (GFP) tag was constructed by pHX-ACP-GFP. The distribution of ACP in "delayed death" was observed by ACP-GFP fusion protein with a confocal microscope. T. gondii ACP transcription and t-ACP expression had no significant decrease in the early 4 h of "delayed death," but there has been a significant decrease in 6 h. The expression of m-ACP had a significant decrease in 4 h which occurred earlier than the t-ACP expression. The number of brightly dot green fluorescence in ACP-GFP mutant decreased with prolonged time. There was very little brightly dot green fluorescence in ACP-GFP mutant when treated with CLDM for 6 h. CLDM could suppress apicoplast proliferation and induce T. gondii "delayed death"; however, it could not directly suppress nucleus coding ACP transcription and expression. T. gondii lacking of apicoplast had a barrier of transit peptide cleavage and t-ACP could not be transformed into m-ACP. The reason for the decrease in ACP expression could be due to excessive t-ACP synthesis in tachyzoites resulting in a negative feedback for the ACP coding gene transcription.

[Overexpression of Toxoplasam gondii ROP18 by Tet-on Lentivirus Expression System].

OBJECTIVE: To construct a 293T mutant cell line over-expressing ROP18 of Toxoplasma gondii by Tet-on lentivirus expression system. METHODS: Rop18 gene of T. gondii was amplified by PCR, and inserted into a lentiviral vector pLVCT-tTR-KRAB. The recombinant plasmid pLVCT-tTR-KRAB-ROP18 (6 µg) and 293T human embryonic kidney cells were co-transfected with psPAX2 (4 µg) and pMD2.G (2 µg) for the packaging. The result of co-transfection was evaluated by fluorescence microscopy. At 48 h and 72 h after co-transfection, the supernatant of the packaging lentivirus was collected for the 293T cell infection. The doxycycline (DOX) was added into the medium to induce the ROP18 expression in 293T cells. The ROP18 fusion expression was observed under fluorescence microscope and detected by RT-PCR after induction. RESULTS: PCR product of the gene fragment encoding ROP18 was 960 bp. The recombinant plasmid pLVCT-tTR-KRAB-ROP18 was identified by PCR and restriction enzyme digestion. Green fluorescence was observed in 293T cells at 48 h post-transfection. Bright green fluorescence was observed in 293T cells at 24 h after DOX induction. RT-PCR results showed that a 960 bp specific band (ROP18 gene) was detectable in 293T cells. CONCLUSION: 293T cell line stably expressing ROP18 is established with Tet-on lentivirus expression system.

[Construction of beta-hydroxyacyl-acyl carrier protein dehydratase mutant of Toxoplasma gondii by tetracycline inducible expression system].

OBJECTIVE: To construct a beta-hydroxyacyl-acyl carrier protein dehydratase (FABZ) mutant of Toxoplasma gondii with tetracycline inducible expression system. METHODS: The fabz gene was amplified from T. gondii genomic DNA, and then used to construct the tetracycline inducible expression vector pTetO7-Sag1-FABZ-Ty-DHFR. The vector was transfected into TATi strain by electroporation. The FABZ defective mutant was selected by pyrimethamine and limitting dilution assay. The expression of Ty-tagged mutant was detected by Western blotting. 5 x 10(5) tachyzoites of FABZ defective mutant were cultured in HFF in the presence of anhydrotetracycline (ATc, 1 microg/ml) for 24 h and 48 h, respectively. The expression of Ty-tagged FABZ protein in the mutant was detected by Western blotting. RESULTS: The mutant could express the transit peptide (t-FABZ) and mature FABZ (m-FABZ) with the Ty-epitope tag. After ATc added in culture medium for 24 h and 48 h, the expression of t-FABZ in the mutant decreased significantly (P<0.05). CONCLUSION: The FABZ mutant is constructed with a tetracycline inducible expression system.

Circular RNA circLIFR suppresses papillary thyroid cancer progression by modulating the miR-429/TIMP2 axis.

PURPOSE: Circular RNAs (circRNAs) are increasingly recognized for their important roles in various cancers, including papillary thyroid cancer (PTC). The specific mechanisms by which the circLIF receptor subunit alpha (circLIFR, hsa_circ_0072309) influences PTC progression remain largely unknown. METHODS: In our study, CircLIFR, miR-429, and TIMP2 levels were assessed using reverse transcription-quantitative PCR. The roles of circLIFR and miR-429 in PTC cells were determined using Cell Counting Kit-8, colony formation, wound healing, and Transwell assays. Western blotting was utilized to examine the levels of TIMP2. The direct interaction between circLIFR, TIMP2, and miR-429 was confirmed using dual-luciferase reporter, RNA immunoprecipitation, and fluorescence in situ hybridization assays. RESULTS: In PTC tissues and cells, a decrease in circLIFR and TIMP2 levels, accompanied by an increase in miR-429 levels, was observed. Overexpression of circLIFR or downregulation of miR-429 effectively suppressed the proliferation and migration of PTC cells. Conversely, the knockdown of circLIFR or overexpression of miR-429 had the opposite effect. Furthermore, circLIFR overexpression suppressed tumor growth in vivo. Mechanistically, circLIFR modulated TIMP2 expression by serving as a sponge for miR-429. Rescue experiments indicated that the antitumor effect of circLIFR could be reversed by miR-429. CONCLUSION: This study confirmed circLIFR as a novel tumor suppressor delayed PTC progression through the miR-429/TIMP2 axis. These findings suggested that circLIFR held promise as a potential therapeutic target for PTC.

[Knock-down of long intergenic noncoding RNA cyclooxygenase 2 (lincRNA-COX2) inhibits apoptosis and polarization into M1 in Listeria monocytogenes-infected macrophages].

Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.

Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems.

In this study, we evaluated four methods to separate and purify Toxoplasma gondii tachyzoites from in vivo and in vitro culture systems, including trypsin digestion, purification with a 3-µm filter, CF-11 cellulose purification, and Percoll purification. Our results indicate that both purification with a 3-µm filter and CF11 cellulose purification methods remove leukocytes or HeLa cells, and can therefore be used as candidate methods for the purification of in vivo and in vitro culture products. Trypsin digestion had a high tachyzoite recovery rate, but 22.35% of leukocytes and 69.64% of HeLa cells remained in the purified products. Percoll solution [30% (v/v)] also had a high tachyzoite recovery rate, but 3.44% of leukocytes and 61.61% of HeLa cells remained in the purified products. The 40% Percoll solution was also a candidate method for purifying tachyzoites from in vivo culture products, with a 65.45% tachyzoite recovery rate and without leukocytes.

The construction and application of a blended teaching model under the strategic background of healthy China.

In order to cultivate the ability of independent learning and lifelong learning of medical students, improve the ability of students to analyze and solve problems, improve the competence of medical talents and cultivate high-level and innovative talents, we have constructed the blended teaching model of "Clinical Case Investigation-Online Open Course Learning-Classroom PBL Seminar-After-Class Health Education". At the same time, an ability-oriented performance evaluation system improved the teaching quality feedback system has also established. This article introduces the construction and application of the blended teaching model, as well as the problems it faces, provides a theoretical basis for the optimization and improvement of this model. It also provides a model theory and practical basis for creating a blended online and offline "golden course" for the professional courses of medical laboratory technology.

Efficacy of ginkgolic acids against Cryptosporidium andersoni in cell culture.

Cryptosporidium is a worldwide waterborne parasite and the treatment is a severe problem in immunocompromised patients. In this study, we used the in vitro culture system to evaluate the anti-Cryptosporidium activity of ginkgolic acids (GAs), nitazoxanide (NTZ), garlicin (GAR), and artemether (ART). The growth of Cryptosporidium andersoni in HCT-8 cells was determined by real-time PCR assay. When exposed to 5.00 µg/ml GAs or 10.00 µg/ml NTZ for 48 h, the number of C. andersoni in cultures was on a very low lever, but the number of parasites did not significantly decrease when exposed to GAR and ART. Our results indicate that GAs could be a potential drug for the treatment of cryptosporidiosis.

[Stable green fluorescent protein expression in Toxoplasma gondii mutant].

OBJECTIVE: To construct a Toxoplasma gondii mutant for stably expressing green fluorescent protein (GFP), and establish method to determine the rate of mutant-infected HeLa cells. METHODS: Freshly lysed-out tachyzoites of T. gondii RH strain were transfected with plasmid ptubP30-GFP/sag-CAT. Stable transformants were selected with chloramphenicol and limited dilution. The expression of GFP in mutant tachyzoite was determined by RT-PCR and fluorescence microscopy. When infected with 1 x 10(4)-1 x 10(7) mutant tachyzoites respectively for 24 h, the total number of HeLa cells with green fluorescence was determined by fluorescent microscope in 10 high-power fields, and the rate of HeLa cells with parasitophorous vacuole was determined by flow cytometry. RESULTS: Untransfected tachy-zoites were killed by chloramphenicol, while the stable transformants showed resistance to chloramphenicol. The expression of GFP gene was detected by RT-PCR. The P30-GFP transfectants displayed fluorescence outside the parasite. The rate of mutant-infected HeLa cells increased with the rise of the number of mutant for infection. When infected with 1 x 10(4)-1 x 10(7) tachyzoites, the numbers of HeLa cells with fluorescence were (14 +/- 6), (133 +/- 45), (332 +/- 93) and (443 +/- 90), and the rates of infected cells were (0.49 +/- 0.09)%, (8.76 +/- 0.50)%, (21.0 +/- 21.49)%, and (39.00 +/- 3.47)% by flow cytometry, respectively. CONCLUSION: T. gondii mutant with GFP tag is constructed, which provides a new method to determine the proliferation when cultured in host cells.

NF-κB/ROS and ERK pathways regulate NLRP3 inflammasome activation in Listeria monocytogenes infected BV2 microglia cells.

Listeria monocytogenes is a food-borne pathogen responsible for neurolisteriosis, which is potentially lethal in immunocompromised individuals. Microglia are the main target cells for L. monocytogenes in central nervous system (CNS). However, the precise mechanisms by which they trigger neuroinflammatory processes remain unknown. The BV2 microglial cell line and a murine model of L. monocytogenes infection were used for experiments in this study. Listeria monocytogenes induced pyroptosis and nucleotide binding and oligomerization, leucine-rich repeat, pyrin domain-containing 3 (NLRP3) inflammasome activation in BV2. Pharmacological inhibition of the NLRP3 inflammasome attenuated L. monocytogenes-induced pyroptosis. Moreover, inhibition of nuclear factor kappa-B (NF-κB) and extracellular regulated protein kinases (ERK) pathways induced a decrease in caspase1 activation and mature IL-1ß-17 secretion. Our collective findings support critical involvement of the NLRP3 inflammasome in L. monocytogenes-induced neuroinflammation and, to an extent, ROS production. In addition, ERK and NF-κB signaling play an important role in activation of the NLRP3 inflammasome, both in vitro and in vivo.

Toxoplasma Gondii Rhoptry Protein 16 (ROP16) Modifies Apoptosis in Human 293T Cells.

Toxoplasma Gondii is an obligate intracellular protozoan. T. gondii tachyzoites can invade nucleated host cells and inhibit their apoptosis. Therefore, the goal of the present study was to determine whether rhoptry protein 16 (ROP16) secreted by the invading T. gondii can reduce the apoptotic response of the host cell. For this purpose, a vector for in vitro overexpression of T. gondii ROP16 was constructed and used to transfect human 293T cells. Cells transfected with the vector robustly expressed ROP16, as evidenced by Western blotting. Apoptosis of 293T cells was induced by incubation with 0.5 µg/mL actinomycin D (ActD) for 24 h, and its magnitude was measured using Annexin V-FITC/PI and TUNEL assays. Cells transfected by ROP16-expressing vector were characterized by a significantly lower level of apoptosis measured by both techniques.Moreover, caspase-3 activity was also reduced. Thus, ROP16 inhibited ActD-induced apoptosis of human 293T cells, documenting the ability of this rhoptry protein to modulate apoptosis of host cells infected by T. gondii.

[TNF-α activates RhoA/ROCK signaling pathway and increases permeability of endothelial cells infected with Listeria monocytogenes].

Objective To investigate the role of Ras homolog gene (Rho) A/Rho-associated coiled-coil containing protein kinase (ROCK) signaling pathway in tumor necrosis factor α (TNF-α) promoting hyper-permeability of vascular endothelial cells infected by Listeria monocytogenes (Lm) . Methods The cultured human umbilical vein endothelial cells (HUVECs) were divided into a control group (uninfected cells), TNF-α treatment group (100 ng/mL TNF-α, for 2 hours), Lm infection group (infected with MOI=10 Lm for 2 hours, then added gentamicin for 0.5 hour), Lm infection and TNF-α treatment group (infected with Lm and then treated with 100 ng/mL TNF-α for 2 hours), and Y-27632 inhibitor group combined with Lm infection and TNF-α treatment (treated with 50 µmol/L ROCK inhibitor Y-27632 for 30 minutes, and then Lm infection and TNF-α treatment as above). The protein levels of RhoA, zonula occluden-1 (ZO-1), occludin and ROCK in HUVECs were detected by Western blot analysis; the permeability of HUVECs was analyzed by the horseradish peroxidase (HRP) leakage; and the distribution of F-actin in HUVECs was detected by fluorescein isothiocyanate (FITC)-labeled phalloidine staining. Results TNF-α reduced the expression of tight junction protein ZO-1 and occludin in Lm-infected HUVECs, promoted its hyper-permeability and cytoskeletal rearrangement, and up-regulated the expression of RhoA and ROCK. ROCK inhibitor Y-27632 obviously inhibited the cytoskeleton rearrangement and hyper-permeability of HUVECs induced by TNF-α. Conclusion TNF-α can enhance hyper-permeability of HUVECs infected by Lm, which may be regulated by RhoA/Rock signaling pathway.

Toxoplasma gondii: a simple Real-time PCR assay to quantify the proliferation of the apicoplast.

A Real-time quantitative PCR assay to quantify the Toxoplasma gondii apicoplast was studied. Primers were designed to amplify a 305bp product specific to T. gondii apicoplast. Standard curves were generated for both T. gondii apicoplast DNA and genomic DNA, and were used to compute the relative concentration of apicoplast DNA copies in the test samples. The results indicated that the copies of T. gondii apicoplast DNA was significantly low when exposed to ciprofloxacin, clindamycin and azithromycin for 48h in the second infectious cycle. Our study shows that the Real-time PCR technique is a simple and quick technique for screening the anti-apicoplast drugs.

Effect of select medium supplements on in vitro development of Cryptosporidium andersoni in HCT-8 cells.

We evaluated the effect of fetal calf serum (FCS), glucose, ascorbic acid, calcium pantothenate, folic acid, and insulin on the growth of Cryptosporidium andersoni in human colon tumor (HCT-8) cells. After being incubated for 48 h, the proliferation of parasites was determined by real-time polymerase chain reaction (PCR) assay, and the development of C. andersoni was observed by transmission electron microscopy (TEM). Ten percent FCS was the best concentration for C. andersoni culture. Glucose, ascorbic acid, and insulin had a significant effect on the growth of C. andersoni when added into 10% FCS RPMI 1640. Calcium pantothenate had no significant effect and folic acid had the inhibited effect. We also observed the stages of trophozoite, macrogamont, microgamont, type I meront, type II meront, and sporozoite of C. andersoni in HCT-8 cells by TEM. Our results indicated that the best medium for C. andersoni was 10% FCS RPMI 1640 medium containing 50 mM glucose, 50 microg/ml ascorbic acid, and 0.3 U/ml insulin. Real-time PCR could provide a quick and precise technique to determine the proliferation of parasites. Cultivation of C. andersoni in HCT-8 cells will facilitate the study of interactions between parasites and host cells as well as provide a reliable system for evaluating anticryptosporidial compound efficacy.

[Real-time PCR in analyzing DNA extraction from Cryptosporidium oocysts].

OBJECTIVE: To compare the quality and quantity of DNA extracted from Cryptosporidium oocysts by different methods. METHODS: Cryptosporidium oocysts were treated with different kinds of lysis buffers from USA Promega (Promega) and Shanghai Generay (Generay) commercial DNA extraction kits, 2% Triton X-100 and 5% guanidine thiocyanate. The oocysts were then broken down by freeze-thawing, proteinase K and sonication. Genomic DNA was purified using the commercial kits or Chelex-100. Real-time PCR technique was used to determine the copies of Cryptosporidium oocyst wall protein (COWP) gene. The Promega commercial DNA extraction kit was used as control. RESULTS: The Promega kit resulted in a higher copy number of COWP gene [(6.45-9.86) x 10(6)] than that of Generay commercial DNA kit [(2.38-3.69) x 10(6)], 5% guanidine thiocyanate [(1.27-21.29) x 10(5)] or 2% Triton X-100 [(2.06-866.70) x 10(3)] , respectively. The method of freeze-thawing plus proteinase K plus sonication provided the highest copy number of COWP gene. CONCLUSION: The method of freeze-thawing + proteinase K + sonication is most effective. The effect of DNA extraction by Generay kit and 5% guanidine thiocyanate is similar to that of Promega kit.