Real-time fluorescence analysis of molecular beacon hybridization during PCR amplification was carried out for all lysates. An isolate was determined to be drug resistant if one of the nine sample wells lacked a characteristic increasing fluorescent signal and the 16S rRNA control molecular beacon present in the same well fluoresced normally. Localization of molecular beacons within their target genes. The accession number of each sequence in the GenBank database is as follows: katG , accession no.
Underlines identify the molecular beacon target sequences. These findings agreed with those of previous genetic investigations. However, when the results for isoniazid resistance were stratified on the basis of sample population, there was a marked heterogeneity in the assay's ability to detect isoniazid resistance.
Stratification on the basis of drug resistance pattern revealed that this disparity was not due to differences among the MDR isolates. A difference was noted among isolates that were resistant only to isoniazid single-drug-resistant isolates. These findings were not a consequence of a limited number of strains that contained unusual mutations.
DNA fingerprinting studies determined that the isoniazid-resistant isolates comprised at least 53 different strains, including at least 9 different strains that were resistant only to isoniazid and that had no identifiable mutations. Multiple mutations were defined as mutations detected in at least two of the three katG , inhA , and ahpC-oxyR amplicons or single mutations detected in the ahpC-oxyR region which do not in themselves confer isoniazid resistance that are associated with additional mutations in katG outside of codon In order to investigate whether mutations in the kasA gene account for additional resistance to isoniazid, we constructed molecular beacons to rapidly screen all of the Madrid and New York M.
No mutations were found in codon 66, , or Ten isolates were found to contain the GS mutation; however, five of these isolates were fully isoniazid susceptible, suggesting that this mutation is not responsible for isoniazid resistance. The five susceptible isolates with GS mutations originated in New York and comprised four different strains, as defined by DNA fingerprinting.
Two of the susceptible kasA strains with the GS mutation were available for repeat susceptibility testing. The molecular beacon assay identified mutations in kasA codon and all katG , inhA , ahpC-oxyR , and rpoB target sequences. The assay was also able to distinguish between wild-type and mutant kasA codon 66, , and sequences when they were tested in control reactions Fig.
These results indicate that all of the molecular beacons were able to detect the appropriate mutations. DNA sequencing of a subset of the isolates that were identified as resistant by the molecular beacon assay confirmed the presence of the expected mutations and deletions. DNA sequencing of the complete katG gene, inhA operon, and ahpC-oxyR genes of two of the isoniazid-resistant isolates from Madrid that were misidentified as isoniazid susceptible by the molecular beacon assay failed to reveal any mutations in these genes.
The single rifampin-resistant isolate that was misidentified as rifampin susceptible originated from an isolate known to consist of a mixture of drug-resistant and drug-susceptible organisms on culture.
Mutational analysis during real-time PCR with molecular beacons complementary to wild-type kasA sequences. We investigated the ability of the results of the molecular beacon assay to be interpreted without real-time analysis. The fluorescence of each molecular beacon at the end of the PCR was measured, and normalized fluorescence intensity values were plotted for each M. Detection of isoniazid-resistant upper panel and rifampin-resistant lower panel M.
The results for each M. The results for the study lysates plus an additional 16 duplicate lysates of isolates from study patients are shown. An isolate is scored as drug susceptible if all of the normalized fluorescence intensity values for that isolate are greater than 0. Isoniazid susceptibility results are plotted for molecular beacons that are specific for katG blue , inhA green , and oxyR-ahpC black and red target amplicons.
Rifampin susceptibility results are plotted for molecular beacons complementary to different target sequences within the core region of the rpoB gene blue, green, black, red, and pink. The colors used to identify the results obtained with each molecular beacon are the same as those used to identify the molecular beacons in Fig. An asterisk identifies the sole rifampin-resistant isolate identified as sensitive by the molecular beacon assay. Understanding the genetic events that lead to drug resistance in clinical M.
The results of this investigation suggest that the frequency of mutations responsible for many cases of isoniazid-resistant tuberculosis may have been incorrectly estimated by prior studies. It suggests that the overall drug resistance profile and, therefore, the clinical history of an M. The differences that were observed between isolates from a reference laboratory and those from a community medical center emphasize the importance of appropriate sample selection for future genetic studies.
Although Molecular beacon analysis for the kasA mutations that were thought to be associated with isoniazid resistance 16 did not improve the sensitivity of the assay. The kasA codon mutations were equally present in isoniazid-susceptible and isoniazid-resistant isolates, and no other kasA mutations were present in any of the isolates tested.
We observed that M. This finding suggests that many clinical isolates develop isoniazid resistance by a stepwise accumulation of mutations, with the first mutation occurring in sequences or genes that have not been commonly associated with resistance.
Multidrug resistance is strongly associated with patients who have had prior treatment for tuberculosis and thereby repeated exposure to isoniazid 5. The accumulated mutations may be important for achieving higher levels of isoniazid resistance or for maintaining full virulence in the human host. This hypothesis does not exclude the possibility that full resistance can also develop in a single step, as is observed in laboratory mutants. However, information on high-level resistance was not available for many of the isolates.
This association will be explored in future studies. This study demonstrates that the use of molecular beacons provides a simple and rapid method for the identification of rifampin-resistant M. Given the increasing worldwide incidence of drug-resistant tuberculosis, the search for assays for the rapid detection of drug resistance has taken on a new sense of urgency.
Unacceptable delays in the diagnosis of drug-resistant tuberculosis can occur when conventional culture-based susceptibility tests are used because of the slow growth of the M. More rapid approaches that use genetic analysis for the detection of mutations associated with drug resistance have been proposed 3 , 4 , 6 , 20 , 23 , However, these methods have restricted applicability to clinical laboratories and are limited principally to the detection of rifampin resistance.
Molecular beacon assays have significant advantages over other techniques. Amplification, molecular beacon hybridization, and analysis are all performed simultaneously in sealed wells. The entire assay, including analysis, can be completed in under 3 h. This was likely due to the closed-well protocol, which eliminates amplicon carryover. With the identification of additional targets for isoniazid resistance, the assay should perform well for the detection of isoniazid resistance in single-drug-resistant isolates.
The molecular beacon assay requires expensive equipment that is not yet widely available. However, it is likely that this type of equipment will become more commonplace in diagnostic laboratories as the broad applicability of closed-tube PCR assays is demonstrated 2 , In this study, drug resistance was predicted equally well by measuring the normalized fluorescence intensities of completed PCRs and by real-time PCR analysis.
These findings suggest that less complex fluorescence viewers that will be able to analyze molecular beacon assays after PCR is completed can be developed. In summary, the molecular mechanisms of isoniazid resistance require further study with representative populations. With the identification of the full complement of resistance mutations, molecular beacon assays will be able to rapidly and simply identify resistance to isoniazid and rifampin, the two principal antituberculosis drugs.
We thank S. If one chooses to initiate, the objective is to control the Target Portal across five Checkpoints. After the 5th Checkpoint, the faction that scored the most points wins the Battle Beacon Challenge.
Battle Beacons can be deployed on almost any Portal. A Battle Beacon can be deployed on a Portal with Fireworks or a Portal Fracker already deployed, because these effects will end prior to the conclusion of the minute battle. Checkpoints occur at 3-minute intervals, with the following escalating Point Values: Point Values increase as the battle progresses to reflect the increasing intensity of the competition, and to create opportunities for a Faction to possibly come from behind.
The maximum Point Value in a single battle is 12 Points. The Faction with the highest total point value successfully captures the Target Portal. After each Checkpoint, the Portal will reverse alignment to the opposing Faction , retaining any Portal Mods applied.
It is merely a treaty among sovereign, independent nations all of whom do whatever they like when their self-interest dictates. Peace, economic prosperity, and political freedom continue to elude most of the world, including citizens of Virginia.
However, a number of great minds after World War Two believed the United Nations like the Articles of Confederation was entirely inadequate. They advocated democratic federal world government for the world. That is correct.
The Earth already has a democratic constitution ready for ratification by the people and nations of the Earth. All that is necessary is that we have the kind of vision for the twenty-first century that our founding fathers had in Philadelphia in The only way out of the chaos and violence engulfing our world today is to have the courage and vision for our planet that the founding fathers had for the early thirteen colonies.
None of the "global crises" faced by our world are soluble under our current world system. Only democratic world government can unify humankind and create institutions that can deal with terrorism, militarism, environmental destruction, population explosion, human rights abuses, and global poverty and misery.
The Constitution for the Federation of Earth , now in twenty-two languages, is designed to create global peace and deal specifically with all these planetary crises. It is time we lived from our vision and courage, not from our fear, timidity, and violence. The only reasonable hope for the future of our children and our planet is ratification of the Constitution and the beginning of a truly human world order based on peace, freedom, and justice rather than fear, violence, and chaos.
The choice is ours, and now, if ever, is the time. The system of domination within the United States and the world is moving toward perfection.
With the advent of the electronic age, and the capacity for computerized tracking of every aspect of the lives of citizens, mechanisms of domination are brought to perfection. The possibilities for privacy, personal dignity, and freedom rapidly recede from memory. Security screening increasingly present for all public functions, public places, or personal travel, is not only designed to humiliate and dehumanize citizens.
It is designed to record electronically all movements of citizens and, ultimately, to condition people to unquestioning obedience, subordination, and acquiescence to governmental authorities.
Currently at U. Citizens must be conditioned to unnecessary double and triple searches until the unquestioned submission to the system of domination becomes second nature. The rationale of security against possible terrorist activity has little to do with the real motivations behind perfecting the system of domination.
The dominators in top echelons of government, military, and the ruling class know perfectly well that their global system of domination and economic exploitation operating for the past five centuries will always spark violent resistance in some people. The exact consequence of their policies occasional violent resistance becomes the proffered rationale for perfecting the system of domination with the electronic apparatus now available under the deceptive heading of "preventing terrorism".
The economic and political system resulting from the collapse of the Middle Ages involved a dynamic confluence of emerging sovereign nation-states and newly borne globalized capitalism.
Nations forming the core of world military and economic power acted to control the wealth-producing process in their own favor. As economist J. Smith labels it, "plunder by raid of the Middle Ages was replaced by plunder by trade. Every nation with power acted to privilege its capitalist class in the system of plunder of the cheap labor and resources of weaker nations in the periphery.
When economic coercion, unequal trade agreements, threats, or manipulation fail to give advantage to the imperial nation, the military is used to destroy all breaks for economic or real political freedom in the periphery. The wealth, resources, and labor power of peripheral nations must not be used for the benefit of peoples in these countries.
Their wealth and resources are forced into a system that feeds the coffers of the rich and powerful, backed by diplomacy and military might within the imperial centers of capital.
The past five centuries is a history of empires, the last of which is the current brutal empire of the United States, ensuring massive starvation and death of exploited peoples throughout the world.
Up to the present, nonviolent civil resistance Gandhi, M. King, Jr. The dominators have viciously suppressed all breaks for freedom in the periphery. They overthrew Mossadeq in Iran in , Arbenz in Guatemala in , massively bombed Vietnam, Cambodia, and Laos in the s, overthrew Sukarno in Indonesia in , supported genocide in East Timor from to , overthrew the Allende government of Chile in , destroyed the Nicaraguan revolution during the s, brutally suppressed resistance in El Salvador and Guatemala in the s and s, destroyed socialism in Yugoslavia in , invaded and occupied Afghanistan in , and did the same in Iraq in They economically blockaded nations trying to act independently of the system of domination, calling them "rogue states" Cuba, North Korea, Libya.
The destruction and occupation of Iraq is only the most recent repression of a nation refusing to lie on its back and open its veins to the vampires in the IMF, World Bank, Washington, DC, and corporate centers of capital. Although resistance worldwide continues, since WW II some twenty-five million people have been slaughtered in wars, most of them civilians, and most of them due to the state terrorism of the imperial centers of power.
By the dawn of the twenty-first century, the global system of imperial sovereign nations inseparable from the global system of capital had solidified five monopolies over world-order that assured their control of the wealth-producing process nearly everywhere.
They solidified monopolies over weapons and military power, over money creation and lending, over private-property laws and rights, over technological mechanisms for the development of wealth, and over communications and information.
Below we will see how a liberated world-order entails breaking each of these monopolies and creating genuine democracy for the first time.
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