Why do nested pcr

This is because nested primers will not find priming sites on any primer dimers or nonspecific artefacts generated in the primary PCR. It is particularly useful for suboptimal nucleic acid samples, such as those extracted from formalin-fixed, paraffin-embedded tissue. Nested PCRs have proven valuable for the detection of microorganisms when they are present in very low quantities.

For example:. This enables […]. This site uses Akismet to reduce spam. Learn how your comment data is processed. News Ticker. Types of PCR. About Acharya Tankeshwar Articles. Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. Early disease confirmation and management is considered very important in disease control of L. Recent emergence of VL and MCL [ 9 , 10 , 11 ], poor treatment response [ 12 ], micro changes within CL profile [ 13 ], widening case distribution [ 8 ], atypical manifestations [ 12 , 13 ], regionally varied risk factors [ 14 , 15 , 16 ] and questionnable potential for visceralization [ 17 ] further necessitates urgent action in this locality.

Detection of all infections and all clinically apparent cases are important. VL essentially requires pre-treatment laboratory confirmation of all cases. Success rate of first line investigation, light microscopy LM depend on parasite load and technical handling though it is the cheapest, least complex, quick and field friendly.

Low parasite counts in chronic and partially treated infections usually lead to false-negative results when traditional diagnostic assays are used. Therefore, more sensitive tools are required to detect those clinically suspected cases that turn negative in LM. In-vitro culturing IVC of parasites is complex, expensive and time consuming while reporting can take time depending on parasite growth in culture media.

All Leishmania inoculations do not end up in successful parasite growth in artificial media. Molecular detection of Leishmania using PCR is the most sensitive method to date. However, molecular techniques are highly expensive, require sophisticated laboratory facilities and require technical expertise limit its usage for performance in resource poor endemic field areas [ 18 ].

However, availability of a highly sensitive PCR assay at a central laboratory would be extremely useful to establish an immediate diagnosis in microscopy negative minority that demands a quick diagnosis. Most researchers have shown PCR as a sensitive and specific tool for diagnosis of leishmaniasis and shown excellent correlations between PCR results and other diagnostic methods such as parasitological and serological tests.

The LAMP is a relatively less complex method that is cost-effective and require low performance time [ 18 ]. But the low sensitivity rate of LAMP limits its use as a diagnostic tool. Although the conventional nested PCR is one of the most sensitive PCR techniques, the possible cross-contaminations and carry over contaminations are the major difficulties associated [ 25 ]. They may occur during opening of reaction tubes during preparation for the second step amplification by transferring amplicons already produced during the first amplification step, subsequently result in false positives and therefore reduce the accuracy of the test.

Therefore it reduces the possibility of cross-contaminations during procedure. Although P and P primers may amplify the other kinetoplastida such as Leptomonas and Crithidia , inner primers P and P were designed as specific for Leishmania genus [ 30 ].

But as shown in Fig. Therefore SN was taken as Therefore P value was taken as about A total of 70 40 patients suspected for CL and 30 patients suspected for VL patients were included in the study. Samples were collected from the patients referred to Department of Parasitology, Faculty of Medicine, University of Colombo after informed written consent. Complete lysis of cells is required to yield a high DNA yield since body tissues have an abundance of contractile proteins, connective tissue and collagen.

Occasional vortexing of the samples was done during the incubation period. Total genomic DNA extracted from a cell pellet of L. For the quality assurance of the new test, negative controls were run parallel to the positive control in each round of PCR.

Few negative controls were used including PCR reactions containing only inner primers, only outer primers, both primers without template DNA and both primers with DNA coming from a negative extraction.

These different negative controls further enhanced the quality of the test by excluding any non-specific amplifications, contaminations of reagents used for DNA extraction and PCR. DNA bands were visualized with an ultraviolet light transilluminator.

Minimum of fields were observed before marked as negative. IVC were observed until completion of two weeks from initial inoculation before marked as negative. A sample was taken as Leishmania positive if it was positive by any one of the control tests performed viz. Reproducibility or repeatability of the assay was determined over one year period by performing a known positive and known negative samples in each round of Mo-STNPCR.

Cost analysis per patient was carried out according to approved guidelines of basic cost accounting for clinical services [ 35 ]. The expenses for laboratory personnel and equipment were not considered for the analysis.

But low positivity of other diagnostic tests as mentioned above confirmed the high sensitivity and accuracy of Mo-STNPCR method compared to those individual diagnostic methods. This study produced a fully sensitive diagnostic tool to detect leishmaniasis while enabling the exclusion of Crithidia spp.

In qPCR, an intercalating dye or fluorescence probe are used for quantifying number of amplified DNA molecules using the strength of signal produced by them. Although qPCR is with high sensitivity and specificity and avoids the requirement for post amplification analysis of PCR, the disadvantages of qPCR which are the high cost, complexity and technically demanding nature limit its usage [ 21 ].

Albeit it can amplify Leishmania species, Leptomonas and Crithidia species [ 30 ]. This is a major disadvantage of the CPCR since Leptomonas and Crithidia can co-exist with Leishmania and leads to misdiagnosis even if they are non-pathogenic to humans [ 37 , 38 ]. In the absence of an indicative skin lesion and in the presence of non-specific clinical features in VL, skin tissue or bone marrow examination needs careful exclusion of these pathogens.

This problem was overcome by designing a new primer pair, P and P which amplified a region between the amplicon of P and P as shown in Fig. The region amplified by P and P is specific only to Leishmania genus and therefore it excludes the amplification of Leptomonas and Crithidia species according to the BLAST search carried out on published GenBank data.

Therefore this method restricted the selection of primers since it required significantly different annealing temperatures [ 42 ]. Also the different research groups tried to physically separate the first and second round amplifications by using different procedures with different materials such as a thin layer of mineral oil, agarose resin and trehalose matrix [ 43 , 44 , 45 ].

But these methods are cumbersome, required the use of specially designed reaction tubes and presence of extraneous materials might interfere with the PCR [ 42 ]. The inner primers showed activity with the positive PCR bands only where tubes were inverted and dissolve the inner primers fixed inside the lid. The monitoring of color change of reaction mixture [the mixture was converted to light blue color after dissolving with inner primers mixed with bromophenol blue ] also re-confirmed the stability of inner primers inside the lid against possible evaporation and condensations.

STNPCR employed in this study has been successfully applied for detection of other pathogenic microorganisms such as Schistosoma mansoni [ 42 , 46 ], Plasmodium falciparum [ 47 ], Yersinia pestis [ 48 ], dengue virus serotypes [ 26 ], Vibrio cholera O1 [ 27 ], Leishmania chagasi [ 25 ], Porcine Circovirus type 2 [ 28 ] and Mycobacterium tuberculosis [ 29 ], causing range of clinical conditions.

Current study used a new combination of previously established inner and outer primers and they were examined using modifications to the technique.

Furthermore, NPCR is likely to have limited use as a diagnostic tool due to carry over and cross-contaminations. However, the selection of a satisfactory primer ratio between inner and outer primers can ensure sufficient reactants until completion of the reaction.

Late presentations, atypical presentations and chronic non-treatment responsive cases in an endemic setting limit the usefulness of clinical detection as well as microscopic or culture detection of leishmaniasis. Even though such scenarios often constitute the minority in an endemic setting, establishing a diagnosis in all cases is necessary.

In such a situation level of sensitivity become more important as compared to the cost of investigation, simplicity or the wider availability. The data supporting the conclusions of this article are included within the article. Additional details are available from the corresponding author on reasonable request. Control of the leishmaniases. Report of a meeting of the WHO expert committee on the control of leishmaniasis, Contract No: Accessed 30 Dec Leishmaniasis: current challenges and prospects for elimination with special focus on the south Asian region.

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