Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

Ekawasti, Fitrine; Cahyaningsih, Umi; Dharmayanti, Ni Luh Putu Indi; Sa’diah, Siti; Subekti, Didik Tulus; Azmi, Zul; Desem, Muhammad Ibrahim

doi:10.14202/ijoh.2021.196-203

Cited by 3 publications

(9 citation statements)

References 47 publications

(68 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The most frequent response in PCR-RFLP is incomplete digestion. In a different instance, Ekawasti et al (2021) also noted that less cutting was present in the GRA1, there was no cutting pattern, and GRA7 fragments were digested by DdeI.…”

Section: Discussionmentioning

confidence: 98%

“…The examination of the restriction fragment length distribution is conducted in silico, or on a computer. When in silico restriction gene programs search for restriction sites, it wraps around the DNA and breaks both strands of the DNA molecule when it encounters a DNA sequence with a shape that matches part of the enzyme, known as a recognition site (Ekawasti et al 2021). Ekawasti et al (2022) use Dde1, HinfI, and Sau961 to differentiate the genotype (strain) of T. gondii.…”

Section: Discussionmentioning

confidence: 99%

“…In silico analysis typically forecasts how they will appear in the experiment. Because the T. gondii genome sequence is publicly available, these methods may be quickly described and optimized using in silico modeling (Endrawati et al 2021;Ekawasti et al 2021Ekawasti et al , 2022. Due to insufficient digestion, in silico prediction analysis and electrophoresis produced different conclusions about the restriction sites.…”

Section: Discussionmentioning

confidence: 99%

“…There are several types of T. gondii strains, including RH (type I), Beverly (type II), Me49 (type II), and C56, VEG (type III) (Liu et al 2015;Sibley et al 2009). The strain of T. gondii can be identified using the restriction fragment length polymorphism (RFLP) pattern, which has been shown to be easy, quick, and reliable (Ekawasti et al 2021).…”

Section: H a Y At I H A Y At Imentioning

confidence: 99%

“…The success of the polymerase chain reaction (PCR), particularly in the field of DNA sequencing, depends significantly on the choice of dense granule primers (GRA1 and GRA7) that are acceptable and suitable for the PCR process. The part of the target DNA sequence to be amplified must be known before amplification is carried out by designing the optimum size primer for the PCR product (Ekawasti et al 2021).…”

Section: H a Y At I H A Y At Imentioning

confidence: 99%

See 4 more Smart Citations

The Patterns of Restriction Fragment of Several Enzymes to Distinguish Toxoplasma gondii Isolates Virulent and Avirulent Strains using GRA1 and GRA7 Genetic Marker

Ekawasti

Purwanto²,

Nepho³

et al. 2023

HAYATI J Biosci

View full text Add to dashboard Cite

Toxoplasma gondii pathogenicity depends on the type derived from a clonal population. A genetic analysis of the locus has been carried out to determine the different genotypes of T. gondii (strain types I, II, and III) that are associated with human toxoplasmosis. The several genotypes of T. gondii (strain types I, II, and III) that are linked to human toxoplasmosis have been identified through genetic study of the locus. In this investigation, PCR-RFLP was found to be a useful, and simple method genotypic characterization. The objective of this study was to genotyping characterize T. gondii RH and BEV strains isolates by PCR-RFLP using several restriction enzymes. T. gondii tachyzoite DNA was extracted and amplified by PCR using dense granule genetic markers (GRA1 and GRA7) designed with Primer3plus. The amplification were digested using the restriction enzymes. The PCR-RFLP amplified dense granule products was used to classify strains into two genotypes of T. gondii (virulent and avirulent). The results demonstrated that the RFLP patterns of the GRA1 and GRA7 gene area digested by DdeI, MvaI, HinfI, RsaI, and Sau96I enzymes can be used to identify virulent or avirulent strains of T. gondii. Toxoplasma gondii RH and BEV strain produced different digestion product which can be used to distinguished the strains.

show abstract

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: H a Y At I H A Y At Imentioning

confidence: 99%

Section: H a Y At I H A Y At Imentioning

confidence: 99%

See 3 more Smart Citations

The Patterns of Restriction Fragment of Several Enzymes to Distinguish Toxoplasma gondii Isolates Virulent and Avirulent Strains using GRA1 and GRA7 Genetic Marker

Ekawasti

Purwanto²,

Nepho³

et al. 2023

HAYATI J Biosci

View full text Add to dashboard Cite

show abstract

In silico restriction site analysis for characterization of Toxoplasma gondii isolate

Ekawasti

Cahyaningsih

Sa’diah

et al. 2022

IOP Conf. Ser.: Earth Environ. Sci.

View full text Add to dashboard Cite

Toxoplasma gondii infection is a serious major public health concern. Toxoplasmosis is a disease that affects humans and warm-blooded animals all over the world. The virulence and severity of the sickness may be influenced by parasite load. The three clonal lineages for biological research are T. gondii genotypes I, II, and III. Using primer genes for gra1, rop1, and mic3, which have been identified as essential proteins for tachyzoite invasion and replication in host cells. PCR was used to determine the genotype of a T. gondii isolate from the Indonesian research center for veterinary science. In silico restriction site analysis was performed on T. gondii isolates using CLC sequence viewer 8.0 software to assess sequence data for the existence of restriction enzyme patterns. Predictable restriction fragment length polymorphism using In silico analysis. T. gondii sequencing genes are In silico digested with different restriction enzymes. The findings show that they can easily distinguish archetypal parasites from biotypes and estimate the genetic diversity of the parasites. According to the interpretation of the data, isolate T. gondii belongs to strain RH genotype I.

show abstract

Optimization of deoxyribonucleic acid extraction and polymerase chain reaction methods for gene detection of Toxoplasma gondii in goat milk

FATMAWATI,

SUWANTI,

MUFASIRIN

et al. 2023

Biodiversitas

View full text Add to dashboard Cite

Abstract. Fatmawati M, Suwanti LT, Mufasirin, Subekti DT, Ekawasti F, Lastuti NDR, Lamid M, Suprihati E, Effendi MH, Al-Arif A. 2023. Optimization of deoxyribonucleic acid extraction and polymerase chain reaction methods for gene detection of Toxoplasma gondii in goat milk. Biodiversitas 24: 5905-5911. Dairy goats (Capra aegagrus hircus) are intermediate hosts that transmit Toxoplasma gondii to humans through goat milk consumption. T. gondii can invade mammary glands and secrete into milk. The method of detecting gene of the T. gondii in milk is through a Polymerase Chain Reaction (PCR) test based on specific genes. The objective of this study was to compare the efficacy methods of the T. gondii molecular assay from the extraction, master mix for PCR assay, and primer that is possible to amplify the gene of T. gondii in goat milk for PCR assay. The research method for purification used flotation using concentrated sugar. This research compared the extraction kit from Kit Extraction 1 (K1), Kit Extraction 2 (K2), and Kit Extraction 3 (K3) with the extraction method listed in the brochure. Meanwhile, for the PCR assay, three types of master mix were used, including Master Mix 1 (M1), Master Mix 2 (M2), and Master Mix 3 (M3). The specific genes used included primers B1, ROP, GRA 1, GRA 7, BAG 1, and P30. Tris borate EDTA (TBE) or Phosphate Buffer Saline Solution (PBS) as buffer milk samples gave the same PCR test results. The results show that the principle of a flotation method test using PBS can be used to prepare the milk sample. The research showed that extraction kit 1 (K1) and extraction kit 2 (K2) gave positive results for the positive control. However, in terms of price, K2 is cheaper than K1. So, to optimize DNA extraction, K2 can be used. The master mix that can be used for PCR testing of milk samples is M1 because M1 gives positive results for positive controls and negative results for negative controls. Primers for optimal results used GRA 7 and B1 with denaturation temperature of 94°C, annealing at 56°C, and an extension of 73°C 33. This research concluded that the type of DNA extraction kit affects the PCR test results for detecting T. gondii in goat milk. It is necessary to optimize the type of master mix to be used in PCR testing and primers capable of amplifying the T. gondii gene.

show abstract

Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

Cited by 3 publications

References 47 publications

The Patterns of Restriction Fragment of Several Enzymes to Distinguish Toxoplasma gondii Isolates Virulent and Avirulent Strains using GRA1 and GRA7 Genetic Marker

The Patterns of Restriction Fragment of Several Enzymes to Distinguish Toxoplasma gondii Isolates Virulent and Avirulent Strains using GRA1 and GRA7 Genetic Marker

In silico restriction site analysis for characterization of Toxoplasma gondii isolate

Optimization of deoxyribonucleic acid extraction and polymerase chain reaction methods for gene detection of Toxoplasma gondii in goat milk

Contact Info

Product

Resources

About